Anti-FGF19 antibodies and methods using same

ABSTRACT

The invention provides anti-FGF19 antibodies, and compositions comprising and methods of using these antibodies, methods using anti-FGF19 antibodies, and methods comprising detection of FGF19 and/or FGFR4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/913,660filed Oct. 27, 2010, which is a divisional of U.S. application Ser. No.12/692,468, filed on Jan. 22, 2010, which is a divisional of U.S.application Ser. No. 11/673,411, filed on Feb. 9, 2007 and issued asU.S. Pat. No. 7,678,373, which claims priority under 35 USC §119 to U.S.Provisional Application No. 60/772,310, filed Feb. 10, 2006, U.S.Provisional Application No. 60/780,608, filed Mar. 9, 2006, and U.S.Provisional Application No. 60/885,866, filed Jan. 19, 2007, the entirecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology. More specifically, the invention concerns anti-FGF19antibodies, uses of same, and detection of FGF19 and/or FGFR4.

BACKGROUND OF THE INVENTION

The fibroblast growth factor (FGF) family is composed of 22 structurallyrelated polypeptides that bind to 4 receptor tyrosine kinases (FGFR1-4)and one kinase deficient receptor (FGFR5) (Eswarakumar et at (2005)Cytokine Growth Factor Rev 16, 139-149; Ornitz et at (2001) Genome Biol2, REVIEWS3005; Sleeman et at (2001) Gene 271, 171-182). FGFs'interaction with FGFR1-4 results in receptor homodimerization andautophosphorylation, recruitment of cytosolic adaptors such as FRS2 andinitiation of multiple signaling pathways (Powers et al (2000) EndocrRelat Cancer 7, 165-197; Schlessinger, J. (2004) Science 306,1506-1507).

FGFs and FGFRs play important roles in development and tissue repair byregulating cell proliferation, migration, chemotaxis, differentiation,morphogenesis and angiogenesis (Ornitz et al (2001) Genome Biol 2,REVIEWS3005; Auguste et al (2003) Cell Tissue Res 314, 157-166; Steilinget al (2003) Curr Opin Biotechnol 14, 533-537). Several FGFs and FGFRsare associated with the pathogenesis of breast, prostate, cervix,stomach and colon cancers (Jeffers et al (2002) Expert Opin Ther Targets6, 469-482; Mattila et al. (2001) Oncogene 20, 2791-2804; Ruohola et al.(2001) Cancer Res 61, 4229-4237; Marsh et al (1999) Oncogene 18,1053-1060; Shimokawa et al (2003) Cancer Res 63, 6116-6120; Jang (2001)Cancer Res 61, 3541-3543; Cappellen (1999) Nat Genet. 23, 18-20;Gowardhan (2005) Br J Cancer 92, 320-327).

FGF19 is a member of the most distant of the seven subfamilies of theFGFs. FGF19 is a high affinity ligand of FGFR4 (Xie et al (1999)Cytokine 11:729-735). FGF19 is normally secreted by the biliary andintestinal epithelium. FGF19 plays a role in cholesterol homeostasis byrepressing hepatic expression of cholesterol-7-α-hydroxylase 1 (Cyp7α1),the rate-limiting enzyme for cholesterol and bile acid synthesis(Gutierrez et al (2006) Arterioscler Thromb Vasc Biol 26, 301-306; Yu etal (2000) J Biol Chem 275, 15482-15489; Holt, J A, et al. (2003) GenesDev 17(130):158). FGF19 ectopic expression in a transgenic mouse modelincreases hepatocytes proliferation, promotes hepatocellular dysplasiaand results in neoplasia by 10 months of age (Nicholes et al. (2002). AmJ Pathol 160, 2295-2307). The mechanism of FGF19 induced hepatocellularcarcinoma is thought to involve FGFR4 interaction. Treatment with FGF-19increases metabolic rate and reverses dietary and leptin-deficientdiabetes. Fu et al (2004) 145:2594-2603. FGF-19 is also described in,for example, Xie et al. (1999) Cytokine 11:729-735; and Harmer et al(2004) 43:629-640.

FGFR4 expression is widely distributed and was reported in developingskeletal muscles, liver, lung, pancreas, adrenal, kidney and brain (Kanet al. (1999) J Biol Chem 274, 15947-15952; Nicholes et al. (2002). Am JPathol 160, 2295-2307; Ozawa et al. (1996) Brain Res Mol Brain Res 41,279-288; Stark et al (1991) Development 113, 641-651). FGFR4amplification was reported in mammary and ovarian adenocarcinomas(Jaakkola et al (1993) Int J Cancer 54, 378-382). FGFR4 mutation andtruncation were correlated with the malignancy and in some cases theprognosis of prostate and lung adenocarcinomas, head and neck squamouscell carcinoma, soft tissue sarcoma, astrocytoma and pituitary adenomas(Jaakkola et al (1993) Int J Cancer 54, 378-382; Morimoto (2003) Cancer98, 2245-2250; Qian (2004) J Clin Endocrinol Metab 89, 1904-1911;Spinola et al. (2005) J Clin Oncol 23, 7307-7311; Streit et al (2004)Int J Cancer 111, 213-217; Wang (1994) Mol Cell Biol 14, 181-188; Yamada(2002) Neurol Res 24, 244-248).

It is clear that there continues to be a need for agents that haveclinical attributes that are optimal for development as therapeuticagents. The invention described herein meets this need and providesother benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention is in part based on the identification of a variety ofFGF19 binding agents (such as antibodies, and fragments thereof). FGF19presents as an important and advantageous therapeutic target, and theinvention provides compositions and methods based on binding FGF19.FGF19 binding agents, as described herein, provide important therapeuticand diagnostic agents for use in targeting pathological conditionsassociated with expression and/or activity of the FGF19-FGFR4 pathways.Accordingly, the invention provides methods, compositions, kits andarticles of manufacture related to FGF19 binding and detection of FGF19and/or FGFR4 binding.

In one aspect, the invention provides an isolated anti-FGF19 antibody,wherein a full length IgG form of the antibody specifically binds humanFGF19 with a binding affinity of about 20 pM or better. In someembodiments, the antibody specifically binds human FGF19 with a bindingaffinity of about 40 pM or better. As is well-established in the art,binding affinity of a ligand to its receptor can be determined using anyof a variety of assays, and expressed in terms of a variety ofquantitative values. Accordingly, in one embodiment, the bindingaffinity is expressed as Kd values and reflects intrinsic bindingaffinity (e.g., with minimized avidity effects). Generally andpreferably, binding affinity is measured in vitro, whether in acell-free or cell-associated setting. Any of a number of assays known inthe art, including those described herein, can be used to obtain bindingaffinity measurements, including, for example, Biacore, radioimmunoassay(RIA) and ELISA.

In one aspect, the invention provides an isolated anti-FGF19 antibody,wherein a full length IgG form of the antibody specifically binds humanFGF19 with a k_(on) of 6×10⁵ (M⁻¹s⁻¹) or better and/or with a K, of5×10⁻⁶ (s⁻¹) or better.

In one aspect, the invention provides an isolated antibody that binds anFGFR4 binding region of FGF19.

In one aspect, the invention provides an isolated antibody that bind apeptide comprising, consisting essentially of or consisting of thefollowing amino acid sequence: GFLPLSHFLPMLPMVPEEPEDLR (SEQ ID NO:9) orHLESDMFSSPLETDSMDPFGLVTGLEAVR (SEQ. ID NO:10).

In some embodiments, the isolated antibody binds a polypeptidecomprising, consisting essentially of or consisting of amino acidnumbers 160-217, 140-159, G133-R155, G156-R180 and/or A183-G192 of themature human FGF19 amino acid sequence (i.e., lacking the signalpeptide). In some embodiments, the isolated antibody binds a polypeptidecomprising, consisting essentially of, or consisting of amino acidnumbers P41-Y47, P41-F58, P51-F58, E81-R88, E124-N132 and/or H164-P171of the mature human FGF19 amino acid sequence (i.e., lacking the signalpeptide).

In one aspect, the invention provides an anti-FGF19 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of: KASQDINSFLS (SEQID NO:1), YRANRLVD (SEQ ID NO:2), LQYDEFPLT (SEQ ID NO:3), TYGVH (SEQ IDNO:5), VIWPGGGTDYNAAFIS (SEQ ID NO:6), and KEYANLYAMDY (SEQ ID NO:7).

In one aspect, the invention provides an anti-FGF19 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of: (a) HVR-L1comprising sequence KASQDINSFLS (SEQ ID NO:1); (b) HVR-L2 comprisingsequence YRANRLVD (SEQ ID NO:2); (c) HVR-L3 comprising sequenceLQYDEFPLT (SEQ ID NO:3); (d) HVR-H1 comprising sequence TYGVH (SEQ IDNO:5); (e) HVR-H2 comprising sequence VIWPGGGTDYNAAFIS (SEQ ID NO:6);and (f) HVR-H3 comprising sequence KEYANLYAMDY (SEQ ID NO:7).

In one aspect, the invention provides an anti-FGF19 antibody comprisinga light chain comprising (a) HVR-L1 comprising sequence KASQDINSFLS (SEQID NO:1); (b) HVR-L2 comprising sequence YRANRLVD (SEQ ID NO:2); and (c)HVR-L3 comprising sequence LQYDEFPLT (SEQ ID NO:3).

In one aspect, the invention provides an anti-FGF19 antibody comprisinga heavy chain comprising

(a) HVR-H1 comprising sequence TYGVH (SEQ ID NO:5); (b) HVR-H2comprising sequence VIWPGGGTDYNAAFIS (SEQ ID NO:6); and (c) HVR-H3comprising sequence KEYANLYAMDY (SEQ ID NO:7).

In one aspect, the invention provides an anti-FGF19 antibody comprising(a) a light chain comprising (i) HVR-L1 comprising sequence KASQDINSFLS(SEQ ID NO:1); (ii) HVR-L2 comprising sequence YRANRLVD (SEQ ID NO:2);and (iii) HVR-L3 comprising sequence LQYDEFPLT (SEQ ID NO:3); and (b) aheavy chain comprising (i) HVR-H1 comprising sequence TYGVH (SEQ IDNO:5); (ii) HVR-H2 comprising sequence VIWPGGGTDYNAAFIS (SEQ ID NO:6);and (iii) HVR-H3 comprising sequence KEYANLYAMDY (SEQ ID NO:7).

In one embodiment, an antibody of the invention comprises a light chainvariable domain having the sequence:

DIKMTQSPSSMYASLGERVTIPCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKVEIKR (SEQ ID NO:4); and comprises a heavychain variable domain having the sequence:

(SEQ ID NO: 8) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWPGGGTDYNAAFISRLSITKDNSKSQVFFKMNSLLANDTAIYFCVRKEYANLYAMDYWGQGTLLTVSA.

In one embodiment, an antibody of the invention comprises a light chaincomprising at least one, at least two or all three of HVR sequencesselected from the group consisting of KASQDINSFLS (SEQ ID NO:1);YRANRLVD (SEQ ID NO:2); and LQYDEFPLT (SEQ ID NO:3).

In one embodiment, the antibody comprises light chain HVR-L1 havingamino acid sequence KASQDINSFLS (SEQ ID NO:1). In one embodiment, theantibody comprises light chain HVR-L2 having amino acid sequenceYRANRLVD (SEQ ID NO:2). In one embodiment, the antibody comprises lightchain HVR-L3 having amino acid sequence LQYDEFPLT (SEQ ID NO:3).

In one embodiment, an antibody of the invention comprises a heavy chaincomprising at least one, at least two or all three of HVR sequencesselected from the group consisting of TYGVH (SEQ ID NO:5); (e)VIWPGGGTDYNAAFIS (SEQ ID NO:6); and KEYANLYAMDY (SEQ ID NO:7).

In one embodiment, the antibody comprises heavy chain HVR-H1 havingamino acid sequence TYGVH (SEQ ID NO:5). In one embodiment, the antibodycomprises heavy chain HVR-H2 having amino acid sequence VIWPGGGTDYNAAFIS(SEQ ID NO:6). In one embodiment, the antibody comprises heavy chainHVR-H3 having amino acid sequence KEYANLYAMDY (SEQ ID NO:7).

In one embodiment, an antibody of the invention comprises a heavy chaincomprising at least one, at least two or all three of HVR sequencesselected from the group consisting of TYGVH (SEQ ID NO:5); (e)VIWPGGGTDYNAAFIS (SEQ ID NO:6); and KEYANLYAMDY (SEQ ID NO:7) and alight chain comprising at least one, at least two or all three of HVRsequences selected from the group consisting of KASQDINSFLS (SEQ ID NO:1); YRANRLVD (SEQ ID NO:2); and LQYDEFPLT (SEQ ID NO:3).

In one embodiment, an antibody of the invention comprises a light chainvariable domain having the sequence:

(SEQ ID NO: 4) DIKMTQSPSSMYASLGERVTIPCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPL TFGAGTKVEIKR.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain having the sequence:

(SEQ ID NO: 8) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWPGGGTDYNAAFISRLSITKDNSKSQVFFICMNSLLANDTAIYFCVRKEYANLYAMDYWGQGILLTVSA.

In another aspect, the invention provides anti-FGF19 monoclonalantibodies that compete with an antibody comprising a light chainvariable domain having the sequence:

DIKMTQSPSSMYASLGERVTIPCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKVEIKR (SEQ ID NO:4) and a heavy chainvariable domain having the sequence:

(SEQ ID NO: 8) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWPGGGTDYNAAFISRLSITKDNSKSQVFFKMNSLLANDTAIYFCV RKEYANLYAMDYWGQGTLLTVSAfor binding to FGF19.

In another aspect, the invention provides anti-FGF19 monoclonalantibodies that bind the same (or a substantially similar) FGF19 epitopeas an antibody comprising a light chain variable domain having thesequence:

DIKMTQSPSSMYASLGERVTIPCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKVEIKR (SEQ ID NO:4) and a heavy chainvariable domain having the sequence:

(SEQ ID NO: 8) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWPGGGTDYNAAFISRLSITKDNSKSQVFFKMNSLLANDTAIYFCVRKEYANLYAMDYWGQGTLLTVSA.

As is known in the art, and as described in greater detail hereinbelow,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below).

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a polyclonal antibody. In some embodiments,the antibody is selected from the group consisting of a chimericantibody, an affinity matured antibody, a humanized antibody, and ahuman antibody. In some embodiments, the antibody is an antibodyfragment. In some embodiments, the antibody is a Fab, Fab′, Fab′-SH,F(ab′)₂, or scFv.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human or humanized sequence (e.g.,framework and/or constant domain sequences). In one embodiment, thenon-human donor is a mouse. In one embodiment, an antigen bindingsequence is synthetic, e.g. obtained by mutagenesis (e.g., phage displayscreening, etc.). In one embodiment, a chimeric antibody of theinvention has murine V regions and human C region. In one embodiment,the murine light chain V region is fused to a human kappa light chain.In one embodiment, the murine heavy chain V region is fused to a humanIgG1 C region.

Humanized antibodies of the invention include those that have amino acidsubstitutions in the FR and affinity maturation variants with changes inthe grafted CDRs. The substituted amino acids in the CDR or FR are notlimited to those present in the donor or recipient antibody. In otherembodiments, the antibodies of the invention further comprise changes inamino acid residues in the Fc region that lead to improved effectorfunction including enhanced CDC and/or ADCC function and B-cell killing.Other antibodies of the invention include those having specific changesthat improve stability. In other embodiments, the antibodies of theinvention comprise changes in amino acid residues in the Fc region thatlead to decreased effector function, e.g. decreased CDC and/or ADCCfunction and/or decreased B-cell killing. In some embodiments, theantibodies of the invention are characterized by decreased binding (suchas absence of binding) to human complement factor Clq and/or human Fcreceptor on natural killer (NK) cells. In some embodiments, theantibodies of the invention are characterized by decreased binding (suchas the absence of binding) to human FcγRI, FcγRIIA, and/or FcγRIIIA. Insome embodiments, the antibodies of the invention is of the IgG class(e.g., IgG1 or IgG4) and comprises at least one mutation in E233, L234,L235, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and/orP329 (numbering according to the EU index). In some embodiments, theantibodies comprise the mutation L234A/L235A or D265A/N297A.

In one aspect, the invention provides anti-FGF19 polypeptides comprisingany of the antigen binding sequences provided herein, wherein theanti-FGF19 polypeptides specifically bind to FGF19.

In one aspect, the invention provides an immunoconjugate(interchangeably termed “antibody drug conjugate” or “ADC”) comprisingany of the anti-FGF19 antibodies disclosed herein conjugated to anagent, such as a drug.

The antibodies of the invention bind FGF19, and in some embodiments, maymodulate one or more aspects of FGF19-associated effects, including butnot limited to FGFR4 activation, FGFR4 downstream molecular signaling,disruption of FGFR4 binding to FGF19, FGFR4 multimerization, expressionof a CYP7α1 gene, phosphorylation of FGFR4, MAPK, FRS2 and/or ERK2,activation of β-catenin, FGF19-promoted cell migration, and/ordisruption of any biologically relevant FGF19 and/or FGFR4 biologicalpathway, and/or treatment and/or prevention of a tumor, cellproliferative disorder or a cancer; and/or treatment or prevention of adisorder associated with FGF19 expression and/or activity (such asincreased FGF19 expression and/or activity). In some embodiments, theantibody of the invention specifically binds to FGF19. In someembodiments, the antibody specifically binds to an FGFR4 binding regionof FGF19. In some embodiments, the antibody specifically binds FGF19with a Kd of about 20 pM or stronger. In some embodiments, the antibodyspecifically binds FGF19 with a Kd of about 40 nM or stronger. In someembodiments, the antibody of the invention reduces, inhibits, and/orblocks FGF19 activity in vivo and/or in vitro. In some embodiments, theantibody competes for binding with FGFR4 (reduces and/or blocks FGFR4binding to FGF19).

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks FGF19-induced repression ofexpression of a CYP7α1 gene in a cell exposed to FGF19.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks FGF19-induced phosphorylation ofFGFR4, MAPK, FRS2 and/or ERK2 in a cell exposed to FGF19.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks FGF19-promoted cell migration. Insome embodiments, the cell is a tumor cell. In some embodiments, thecell is a tumor cell. In some embodiments, the cell is an HCT116 cell.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks Wnt pathway activation in a cell.In some embodiments, Wnt pathway activation comprises one or more ofβ-catenin immunoreactivity, tyrosine phosphorylation of β-catenin,expression of Wnt target genes, β-catenin mutation, and E-cadherinbinding to β-catenin. Detection of Wnt pathway activation is known inthe art, and some examples are described and exemplified herein.

In one aspect, the invention provides compositions comprising one ormore antibodies of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In another aspect, the invention supplies a composition comprising oneor more anti-FGF19 antibodies described herein, and a carrier. Thiscomposition may further comprise a second medicament, wherein theantibody is a first medicament. This second medicament, for cancertreatment, for example, may be another antibody, chemotherapeutic agent,cytotoxic agent, anti-angiogenic agent, immunosuppressive agent,prodrug, cytokine, cytokine antagonist, cytotoxic radiotherapy,corticosteroid, anti-emetic cancer vaccine, analgesic, anti-vascularagent, or growth-inhibitory agent. In another embodiment, a secondmedicament is administered to the subject in an effective amount,wherein the antibody is a first medicament. This second medicament ismore than one medicament, and is preferably another antibody,chemotherapeutic agent, cytotoxic agent, anti-angiogenic agent,immunosuppressive agent, prodrug, cytokine, cytokine antagonist,cytotoxic radiotherapy, corticosteroid, anti-emetic, cancer vaccine,analgesic, anti-vascular agent, or growth-inhibitory agent. Morespecific agents include, for example, irinotecan (CAMPTOSAR®), cetuximab(ERBITUX®), fulvestrant (FASLODEX®), vinorelbine (NAVELBINE®),EFG-receptor antagonists such as erlotinib (TARCEVA®) VEGF antagonistssuch as bevacizumab (AVASTIN®), vincristine (ONCOVIN®), inhibitors ofmTor (a serine/threonine protein kinase) such as rapamycin and CCI-779,and anti-HER1, HER2, ErbB, and/or EGFR antagonists such as trastuzumab(HERCEPTIN®), pertuzumab (OMNITARG™), or lapatinib, and other cytotoxicagents including chemotherapeutic agents. In some embodiments, thesecond medicament is an anti-estrogen drug such as tamoxifen,fulvestrant, or an aromatase inhibitor, an antagonist to vascularendothelial growth factor (VEGF) or to ErbB or the Efb receptor, orHer-1 or Her-2. In some embodiments, the second medicament is tamoxifen,letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant,vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib,lapatinib, or trastuzumab, and preferably, the second medicament iserlotinib, bevacizumab, or trastuzumab.

In one aspect, the invention provides an anti-idiotype antibody thatspecifically binds an anti-FGF19 antibody of the invention.

In one aspect, the invention provides nucleic acids encoding ananti-FGF19 antibody of the invention.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides compositions comprising one ormore nucleic acid of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods of making an antibody ofthe invention. For example, the invention provides methods of making ananti-FGF19 antibody (which, as defined herein includes full length andfragments thereof), said method comprising expressing in a suitable hostcell a recombinant vector of the invention encoding said antibody, andrecovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more anti-FGF19antibodies of the invention. In one embodiment, the compositioncomprises a nucleic acid of the invention. In one embodiment, acomposition comprising an antibody further comprises a carrier, which insome embodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (for e.g., the antibody) to anindividual (such as instructions for any of the methods describedherein).

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more anti-FGF19 antibodies ofthe invention; and a second container comprising a buffer. In oneembodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antibody further comprises acarrier, which in some embodiments is pharmaceutically acceptable. Inone embodiment, a kit further comprises instructions for administeringthe composition (for e.g., the antibody) to an individual.

In one aspect, the invention provides use of an anti-FGF19 antibody ofthe invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a cancer, a tumor,and/or a cell proliferative disorder. In some embodiments, the cancer, atumor, and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the cancer, a tumor,and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the cancer, a tumor,and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the cancer, a tumor,and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a cancer, a tumor,and/or a cell proliferative disorder. In some embodiments, the cancer, atumor, and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disorder, such as a cancer, a tumor, and/or a cellproliferative disorder. In some embodiments, the cancer, a tumor, and/ora cell proliferative disorder is colorectal cancer, hepatocellularcarcinoma, lung cancer, breast cancer, or pancreatic cancer. In someembodiments, the disorder is a liver disorder, such as cirrhosis. Insome embodiments, the disorder is a wasting disorder.

The invention provides methods and compositions useful for modulatingdisease states associated with expression and/or activity of FGF19and/or FGFR4, such as increased expression and/or activity or undesiredexpression and/or activity, said methods comprising administration of aneffective dose of an anti-FGF19 antibody to an individual in need ofsuch treatment.

In one aspect, the invention provides methods for killing a cell (suchas a cancer or tumor cell), the methods comprising administering aneffective amount of an anti-FGF19 antibody to an individual in need ofsuch treatment.

In one aspect, the invention provides methods for reducing, inhibiting,blocking, or preventing growth of a tumor or cancer, the methodscomprising administering an effective amount of an anti-FGF19 antibodyto an individual in need of such treatment.

Methods of the invention can be used to affect any suitable pathologicalstate. Exemplary disorders are described herein, and include a cancerselected from the group consisting of esophageal cancer, bladder cancer,lung cancer, ovarian cancer, pancreatic cancer, mammary fibroadenoma,prostate cancer, head and neck squamous cell carcinoma, soft tissuesarcoma, astrocytoma, pituitary cancer, breast cancer, neuroblastomas,melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC),epithelial carcinomas, brain cancer, endometrial cancer, testis cancer,cholangiocarcinoma, gallbladder carcinoma, and hepatocellular carcinoma.

In one embodiment, a cell that is targeted in a method of the inventionis a cancer cell. For example, a cancer cell can be one selected fromthe group consisting of a breast cancer cell, a colorectal cancer cell,a lung cancer cell, a papillary carcinoma cell, a colon cancer cell, apancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell,a central nervous system cancer cell, an esophageal cancer cell, anosteogenic sarcoma cell, a renal carcinoma cell, a hepatocellularcarcinoma cell, a bladder cancer cell, a gastric carcinoma cell, a headand neck squamous carcinoma cell, a melanoma cell, a leukemia cell, abrain cancer cell, a endometrial cancer cell, a testis cancer cell, acholangiocarcinoma cell, a gallbladder carcinoma cell, a lung cancercell, and/or a prostate cancer cell. In one embodiment, a cell that istargeted in a method of the invention is a hyperproliferative and/orhyperplastic cell. In one embodiment, a cell that is targeted in amethod of the invention is a dysplastic cell. In yet another embodiment,a cell that is targeted in a method of the invention is a metastaticcell.

In one embodiment of the invention, the cell that is targeted is acirrhotic liver cell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (for e.g., a cancer cell) isexposed to radiation treatment or a chemotherapeutic agent.

Any suitable anti-FGF19 antibody may be used for methods involvingtreatment and/or prevention of a disorder, including monoclonal and/orpolyclonal antibodies, a human antibody, a chimeric antibody, anaffinity-matured antibody, a humanized antibody, and/or an antibodyfragment. In some embodiments, the anti-FGF19 antibody is any of theanti-FGF19 antibodies described herein.

In another aspect, the invention provides a complex of any of theanti-FGF19 antibodies described herein and FGF19. In some embodiments,the complex is in vivo or in vitro. In some embodiments, the anti-FGF19antibody is detectably labeled.

In another aspect, the invention provides methods for detection ofFGF19, the methods comprising detecting FGF19-anti-FGF19 antibodycomplex in a biological sample. The term “detection” as used hereinincludes qualitative and/or quantitative detection (measuring levels)with or without reference to a control.

In another aspect, the invention provides methods for detecting adisorder associated with FGF19 expression and/or activity, the methodscomprising detecting FGF19 in a biological sample from an individual. Insome embodiments, the FGF19 expression is increased expression orabnormal expression. In some embodiments, the disorder is a tumor,cancer, and/or a cell proliferative disorder, such as colorectal cancer,lung cancer, hepatocellular carcinoma, breast cancer and/or pancreaticcancer. In some embodiment, the biological sample is serum or of atumor.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGFR4 expression and/or activity, the methodscomprising detecting FGFR4 in a biological sample from an individual. Insome embodiments, FGFR4 expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as colorectal cancer, lung cancer,hepatocellular carcinoma, breast cancer and/or pancreatic cancer. Insome embodiment, the biological sample is serum or of a tumor.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGFR4 and FGF19 expression and/or activity, themethods comprising detecting FGFR4 and FGF19 in a biological sample froman individual. In some embodiments, the FGF19 expression is increasedexpression or abnormal expression. In some embodiments, FGFR4 expressionis increased expression or abnormal expression. In some embodiments, thedisorder is a tumor, cancer, and/or a cell proliferative disorder, suchas colorectal cancer, lung cancer, hepatocellular carcinoma, breastcancer and/or pancreatic cancer. In some embodiment, the biologicalsample is serum or of a tumor. In some embodiments, expression of FGFR4is detected in a first biological sample, and expression of FGF19 isdetected in a second biological sample.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGF19expression, if any, in an individual's biological sample; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGF19 expression detected instep (a). In some embodiments, increased FGF19 expression in theindividual's biological sample relative to a reference value or controlsample is detected. In some embodiments, decreased FGF19 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected in the individual. In some embodiments, FGF19expression is detected and treatment with an anti-FGF19 antibody isselected. In some embodiments, the individual has a tumor, cancer,and/or a cell proliferative disorder, such as colorectal cancer, lungcancer, hepatocellular carcinoma, breast cancer and/or pancreaticcancer.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGFR4expression, if any, in an individual's biological sample; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGFR4 expression detected instep (a). In some embodiments, increased FGFR4 expression in theindividual's biological sample relative to a reference value or controlsample is detected. In some embodiments, decreased FGFR4 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected in the individual. In some embodiments, FGFR4expression is detected and treatment with an anti-FGF19 antibody isselected. In some embodiments, the individual has a tumor, cancer,and/or a cell proliferative disorder, such as colorectal cancer, lungcancer, hepatocellular carcinoma, breast cancer and/or pancreaticcancer.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGF19and FGFR4 expression, if any, in an individual's biological sample; and(b) subsequence to step (a), selecting treatment for the individual,wherein the selection of treatment is based on the FGF19 and FGFR4expression detected in step (a). In some embodiments, increased FGF19expression in the individual's biological sample relative to a referencevalue or control sample is detected. In some embodiments, decreasedFGF19 expression in the individual's biological sample relative to areference value or control sample is detected in the individual. In someembodiments, increased FGFR4 expression in the individual's biologicalsample relative to a reference value or control sample is detected. Insome embodiments, decreased FGFR4 expression in the individual'sbiological sample relative to a reference value or control sample isdetected in the individual. In some embodiments, FGFR4 and FGF19expression are detected and treatment with an anti-FGF19 antibody isselected. In some embodiments, expression of FGFR4 is detected in afirst biological sample, and expression of FGF19 is detected in a secondbiological sample. In some embodiments, the individual has a tumor,cancer, and/or a cell proliferative disorder, such as colorectal cancer,lung cancer, hepatocellular carcinoma, breast cancer and/or pancreaticcancer.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, furtherwherein FGF19 expression and/or FGFR4 expression is detected in theindividual's biological sample before, during or after administration ofan anti-FGF19 antibody. In some embodiments, the biological sample is ofthe cancer, tumor and/or cell proliferative disorder. In someembodiments, the biological sample is serum. In some embodiments, FGF19over-expression is detected before, during and/or after administrationof an anti-FGF19 antibody. In some embodiments, FGFR4 expression isdetected before, during and/or after administration of an anti-FGF19antibody. Expression may be detected before; during; after; before andduring; before and after; during and after; or before, during and afteradministration of an anti-FGF19 antibody.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, wherein abiological sample of the cancer, tumor and/or cell disorder or liverdisorder expresses FGF19 and/or FGFR4.

In embodiments involving detection, expression of FGFR4 downstreammolecular signaling may be detected in addition to or as an alternativeto detection of FGFR4 expression. In some embodiments, detection ofFGFR4 downstream molecular signaling comprises one or more of detectionof phosphorylation of MAPK, FRS2 or ERK2.

In some embodiments involving detection, expression of FGFR4 comprisesdetection of FGFR4 gene deletion, gene amplification and/or genemutation. In some embodiments involving detection, expression of FGF19comprises detection of FGF19 gene deletion, gene amplification and/orgene mutation.

Some embodiments involving detection further comprise detection of Wntpathway activation. In some embodiments, detection of Wnt pathwayactivation comprises one or more of tyrosine phosphorylation ofβ-catenin, expression of Wnt target genes, β-catenin mutation, andE-cadherin binding to β-catenin. Detection of Wnt pathway activation isknown in the art, and some examples are described and exemplifiedherein.

In some embodiments, the treatment is for a cancer selected from thegroup consisting of colorectal cancer, lung cancer, ovarian cancer,pituitary cancer, pancreatic cancer, mammary fibroadenoma, prostatecancer, head and neck squamous cell carcinoma, soft tissue sarcoma,breast cancer, neuroblastomas, melanoma, breast carcinoma, gastriccancer, colorectal cancer (CRC), epithelial carcinomas, brain cancer,endometrial cancer, testis cancer, cholangiocarcinoma, gallbladdercarcinoma, and hepatocellular carcinoma.

Biological samples are described herein, e.g., in the definition ofBiological Sample. In some embodiment, the biological sample is serum orof a tumor.

In embodiments involving detection of FGF19 and/or FGFR4 expression,FGF19 and/or FGFR4 polynucleotide expression and/or FGF19 and/or FGFR4polypeptide expression may be detected. In some embodiments involvingdetection of FGF19 and/or FGFR4 expression, FGF19 and/or FGFR4 mRNAexpression is detected. In other embodiments, FGF19 and/or FGFR4polypeptide expression is detected using an anti-FGF19 agent and/or ananti-FGFR4 agent. In some embodiments, FGF19 and/or FGFR4 polypeptideexpression is detected using an antibody. Any suitable antibody may beused for detection and/or diagnosis, including monoclonal and/orpolyclonal antibodies, a human antibody, a chimeric antibody, anaffinity-matured antibody, a humanized antibody, and/or an antibodyfragment. In some embodiments, an anti-FGF19 antibody described hereinis used for detection. In some embodiments, FGF19 and/or FGFR4polypeptide expression is detected using immunohistochemistry (IHC). Insome embodiments, FGF19 expression is scored at 2 or higher using anIHC.

In some embodiments involving detection of FGF19 and/or FGFR4expression, presence and/or absence and/or level of FGF19 and/or FGFR4expression may be detected. FGF19 and/or FGFR4 expression may beincreased. It is understood that absence of FGF19 and/or FGFR4expression includes insignificant, or de minimus levels. In someembodiments, FGF19 expression in the test biological sample is higherthan that observed for a control biological sample (or control orreference level of expression). In some embodiments, FGF19 expression isat least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 75-fold, 100-fold, 150-fold higher, or higher, in the testbiological sample than in the control biological sample. In someembodiments, FGF19 polypeptide expression is determined in animmunohistochemistry (“IHC”) assay to score at least 2 or higher forstaining intensity. In some embodiments, FGF19 polypeptide expression isdetermined in an IHC assay to score at least 1 or higher, or at least 3or higher for staining intensity. In some embodiments, FGF19 expressionin the test biological sample is lower than that observed for a controlbiological sample (or control expression level).

In one aspect, the invention provides an isolated polynucleotidecomprising, consisting of, or consisting essentially of one or more ofthe following polynucleotide sequences:

(SEQ ID NO: 41) GAT CCC CCC TCG TGA GTC TAG ATC TAT TCA AGA GATAGA TCT AGA CTC ACG AGG TTT TTT GGA AA; (SEQ ID NO: 42)AGC TTT TCC AAA AAA CCT CGT GAG TCT AGA TCT ATCTCT TGA ATA GAT CTA GAC TCA CGA GGG GG; (SEQ ID NO: 43)GAT CCC CGA ACC GCA TTG GAG GCA TTA TCA AGA GAAATG CCT CCA ATG CGG TTC TTT TTT GGA AA; or (SEQ ID NO: 44)AGC TTT TCC AAA AAA GAA CCG CAT TGG AGG CAT TTCTCT TGA TAA TGC CTC CAA TGC GGT TCG GG.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B: depicts the amino acid sequences of mouse anti-humanFGF19 monoclonal antibody 1A6 variable regions. (A) light chain variableregion (SEQ ID NO:4). (B) heavy chain variable region (SEQ ID NO:8)Amino acids are numbered according to Kabat. The positions of the HVRsare depicted in the Figures.

FIG. 2: FGF19 and FGFR4 mRNA expression in normal and tumor samples fromcolon (A) and lung (B) were evaluated by semi quantitative RT-PCR.Values were normalized using GAPDH expression and compared to the lowestexpressing sample for each tissue. Representative images of bright-field(left panels) and dark-field (right panels) illumination of in situhybridization with FGF19 and FGFR4 riboprobes and FGF19immunohistochemistry (IHC) (lower panels) in colon adenocarcinoma (C),lung squamous cell carcinoma (D), and hepatocellular carcinoma (E).Representative images of bright-field (top left panel) and dark-field(top right panels) illumination of in situ hybridization with the FGF19riboprobe and FGF19 IHC (lower panels) in cirrhotic liver nodules (F).

FIG. 3: FGF19 and FGFR4 mRNA and protein expression in tumor cell linesand xenograft tissue. (A) FGF19 and FGFR4 mRNA had high relativeexpression in colon tumor cell lines, Colo205, SW620, and HCT116. FGF19and FGFR4 mRNA expression were evaluated using semi-quantitative RT-PCR.Values were normalized using GAPDH expression and compared to the lowestexpressing cell line for each gene. (B) Western blot of FGF19 and FGFR4protein expressed in a panel of colon cancer cell lines. FGF19 proteinexpression was confirmed in colon cancer cell lines byimmunoprecipitation and western blot analysis. (C), (D) A subset ofcolon cancer cell lines was inoculated subcutaneously in nude mice.After 3 weeks, tumors were excised and processed for FGF19immunostaining. Pictures of representative fields at 400× (C) and 400×with an inset of 600× (bar=25 μm) (D) are shown.

FIG. 4: FGF19 bound to components of the FGFR4 complex. (A) FGF19 andFGFR4 protein were incubated with microwell adsorbed heparin sulfateproteoglycan. The binding was detected with biotinylated specificantibodies followed by horseradish peroxydase conjugated streptavidineand colorimetric substrate. (B) FGF19 and FGF 1 protein were incubatedwith heparin-agarose and the gel slurry was washed with 1 ml bindingbuffer containing various NaCl concentrations. The remainingheparin-agarose bound proteins were eluted with SDS PAGE sample bufferand analyzed by Western blot. The control lane (Cont.) represented theoriginal amount of protein directly loaded on the gel. FGF19 protein hada unique binding specificity for FGFR4 captured to a solid phase (C) orin solution (D). (E) FGF19 protein binding to solid phase capturedFGFR4-Fc protein in the presence of by various glycosaminoglycans (HepSulf: heparin sulfate; CS B: condtroitin sulfate B; CS A: chondroitinsulfate A; CS b: chondroitin sulfate C). (F) FGF19 protein binding tosolid phase captured FGFR4 IgG protein in the presence of heparinfragments of various lengths. (G) Scatchard analysis of ¹²⁵I-FGF19protein binding to solid phase captured FGFR4-Fc protein.

FIG. 5: Anti-FGF19 monoclonal antibody 1A6 inhibited FGF19 biologicalactivities in vitro. (A) Binding of monoclonal antibodies to solid phasecaptured FGF19 protein. (B) Binding of FGF19 protein to solid phasecaptured FGFR4-Fc protein in the presence of anti-FGF19 monoclonalantibodies (“mabs”). (C) Treatment with anti-FGF19 mab 1A6 inhibitedFGF19 activation of a FGF signaling pathway. (D) Treatment withanti-FGF19 mab 1A6 inhibited FGF19-induced CYP7α1 repression. HEPG2cells incubated overnight in serum free medium were treated with FGF19protein in the presence or absence of antibodies. CYP7α1 expression wasevaluated by semi-quantitative RT-PCR using gene specific primers andprobes and normalized to GAPDH expression. (E) Treatment with anti-FGF19mab 1A6 inhibited FGF19-promoted HCT116 cell migration. The surface of 8μm porosity 24-well modified Boyden chambers was coated with type 1collagen. Cells in serum free medium were added to the upper chamber.Cells that migrated to the lower chamber following addition of the samemedia containing FGF19 and various concentrations of anti-FGF19 mab 1A6were stained and counted. Triplicate sets of data were averaged for eachcondition.

FIG. 6: Identification of epitopes recognized by anti-FGF19 antibodies.FGF19 protein was incubated for 2 h with agarose coupled antibody. Theresin was washed and digested with trypsin overnight at 37 C. The gelslurry was washed and the bound peptides were eluted and analyzed byMALDI-TOF-MS. (A) Mass spectroscopic analysis of soluble fraction oftrypsin digested FGF19 bound to agarose coupled mab 1A6. (B) Massspectroscopic analysis of FGF19 tryptic peptide eluted from agarosecoupled 1A6 antibody. (C) Mass spectroscopic analysis of FGF19 trypticpeptide eluted from agarose coupled 1A 1 antibody. (D) Peptidecompetition of anti-FGF19 mab 1D1 binding to solid phase-captured FGF19.Anti-FGF19 mab 1D1 was incubated with FGF19-His captured to nickelcoated plates in the presence of peptides representing various portionsof FGF19 protein. The antibody binding was detected with aHRP-conjugated anti-mouse IgG and chromogenic substrate. (E) Mapping of1A6 epitope (indicated with an arrow) onto the FGF19-FGFR4 interactionmodel. FGFR4 surface is represented as a globular form whereas FGF19 isrepresented as a ribbon.

FIG. 7: Treatment with anti-FGF19 mouse monoclonal antibody 1A6inhibited colon tumor growth in vivo. Athymic mice were subcutaneouslyinoculated with 5×10⁶ HCT1126 or Colo201 cells. Mice bearing establishedtumors of equivalent volume (˜100 mm³) were randomized into groups andtreated intraperitoneally twice weekly with anti-FGF19 mab 1A6 or acontrol antibody. Results are given as mean tumor volume+/−sem. At theend of the studies, HCT116 xenograft tumors and Colo201 xenograft tumorsfrom anti-FGF19 mab 1A6-treated or control antibody-treated mice wereexcised, homogenized and analyzed for FGFR4, FRS2, ERK and β-cateninactivation by Western blot. (A) Growth of HCT116 colon tumor xenograftswas inhibited by treatment with anti-FGF19 mab 1A6 compared to treatmentwith control antibody. (B) Phosphorylation of FGFR4, FRS2, and ERK, andβ-catenin activation was inhibited in anti-FGF19 mab 1A6-treated HCT116xenograft tumors. (C) Growth of Colo201 colon tumor xenografts wasinhibited by treatment with anti-FGF19 mab 1A6 compared to treatment ofcontrol antibody. (D) Phosphorylation of FGFR4, FRS2, and ERK, andβ-catenin activation was inhibited in anti-FGF19 mab 1A6-treated Colo201xenograft tumors. For (A) and (C), arrows indicate administration oftreatment. Results are given as mean tumor volume±SE.

FIG. 8: Antibody epitope sequencing. Collision induced dissociation andmanual sequencing of peptides isolated using an epitope excisionprocedure. (A) Sequence of a peptide comprising an epitope of anti-FGF19mab 1A6 (SEQ ID NO:9). (B) Sequence of a peptide comprising an epitopeof anti-FGF19 mab 1A1 (SEQ ID NO:10).

FIG. 9: Treatment with anti-FGF19 mouse monoclonal antibody 1A6inhibited hepatocellular carcinoma in vivo in a FGF19-transgenichepatocellular carcinoma animal model. FGF19 transgenic mice weretreated with either a control antibody or anti-FGF19 mab 1A6, and liverwas collected for gross evaluation (A). MicroCT analysis using aniodinated triglyceride for enhancement of normal hepatocytesdemonstrated increased unenhanced tumor volume in control treated versusanti-FGF19 mab 1A6-treated liver (B).

FIG. 10: Treatment with FGF19 protein promoted HCT116 cell migration.The surface of 8 μm porosity 24-well modified Boyden chambers was coatedtype 1 collagen. HCT116 cells in serum free medium were added to theupper chamber. The lower chamber was filled with the same mediacontaining various concentrations of FGF19 and the plates were incubatedat 37° C. The next day the cells that migrated to the lower side of theinsert were stained and counted under a microscope. Triplicate sets ofdata were averaged for each condition.

FIG. 11: Treatment with FGF19 induced tyrosine phosphorylation ofβ-catenin and caused loss of E-cadherin binding to β-catenin in coloncancer cell line HCT116. Serum-starved colon cancer cells were treatedwith either vehicle (“veh”) (control) or FGF19 at 25 and 100 ng/ml for10 minutes. Tyrosine phosphorylation of β-catenin was determined byimmunoprecipitation (“IP”) and immunoblotting (“WB”). The same blot wasstripped and reprobed using an anti-E-cadherin antibody and subsequentlyreprobed for total β-catenin using an anti-β-catenin antibody.Representative blots from three separate experiments are shown.Quantitative analysis of β-catenin phosphorylation and β-catenin boundto E-cadherin was determined by calculating the ratio between thephosphorylated E-cadherin and total β-catenin levels from three separateexperiments (mean values±SE).

FIGS. 12A and B: Treatment with anti-FGF19 antibody reducedactive-β-catenin levels in HCT116 cells. Cells were grown in thepresence of serum and treated with either control (gp120) or anti-FGF19mab 1A6 (both at 20 μg/ml) for varying time intervals. (A)Active-β-catenin (“act-β-cat”) levels (N-terminally dephosphorylatedβ-catenin) were determined by immunoblotting. The same blot was strippedand reprobed for total β-catenin (“β-cat”) levels. Representative blotsfrom three separate experiments are shown. (B) Quantitative analysis ofactive-β-catenin levels at 24 hrs post-treatment as determined bycalculating the ratio between the active-β-catenin and total β-cateninlevels from three separate experiments (mean values±SE). β-actin levelswere determined as a control.

FIG. 13: Treatment with anti-FGF19 antibody induced phosphorylation onβ-catenin amino acid residues Ser33/Ser37/Ser45 and Thr41 in HCT116cells. Cells were grown in the presence of serum and treated with MG132(1 μM) for 4 hrs followed by treatment with either control (gp120) oranti-FGF19 (1A6) antibody (both 20 μg/ml) for 24 hrs. β-cateninphosphorylation on Ser33/S37/S45 and Thr41 was analyzed byimmunoblotting. The same blot was stripped and reprobed for totalβ-catenin. Representative blots from three separate experiments areshown. β-actin levels were determined as a control.

FIG. 14: Treatment with FGFR4-directed shRNA suppressed expression ofFGFR4 protein and active-β-catenin in HCT116 cells. FGFR4 knockdownvectors were constructed by designing and cloning shRNA sequences intoan retroviral inducible expression vector system. The cDNAs weretransfected and stable cell lines expressing siRNA were generated inHCT116 cells using puromycin selection. Stable cell lines comprisingcontrol shRNA and FGFR4-directed shRNA were treated with or withoutdoxycycline (Dox) and FGFR4 protein and active-β-catenin levels weredetermined by immunoprecipitation and immunoblotting. Representativeblots from three separate experiments are shown.

FIG. 15: Indirect quantification of N-terminal β-catenin phosphorylationlevels using linear ion trap mass spectrometry. Data dependent tandemmass spectrometry on N-terminal peptide from immunoprecipitatedβ-catenin from cells treated with MG132 followed by control (gp120) oranti-FGF19 mab 1A6 was performed using a linear ion trap instrument asdescribed in the Examples. Cross-correlation scores for each CIDspectrum were generated and the relative abundance of peptides wasdetermined. The data were normalized to the signal intensities of othernon-related peptides that showed no difference in signal intensitiesfrom the treated and untreated samples.

FIG. 16: Treatment with anti-FGF19 antibody reduced Wnt-target genetranscription levels in colon cancer cells. HCT116 cell were grown inthe presence of serum and treated with either control (gp120) or 1A6antibody (20 μm') for 6 hrs. β-catenin target gene (cyclin D1, CD44,E-cadherin, and c-jun) expression levels were analyzed by Taqmananalysis. Analyses of data were performed using Sequence Detector 1.6.3(PE Applied Biosystems) and results were normalized to RPL19 geneexpression level.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein provides anti-FGF19 antibodies, which are usefulfor, e.g., treatment or prevention of disease states associated withexpression and/or activity of FGF19, such as increased expression and/oractivity or undesired expression and/or activity. In some embodiments,the antibodies of the invention are used to treat a tumor, a cancer,and/or a cell proliferative disorder.

In another aspect, the anti-FGF19 antibodies of the invention findutility as reagents for detection and/or isolation of FGF19, such asdetention of FGF19 in various tissues and cell type.

The invention further provides methods of making anti-FGF19 antibodies,polynucleotides encoding anti-FGF19 antibodies, and cells comprisingpolynucleotides encoding anti-FGF19 antibodies.

In another aspect, the invention provides methods comprising detectionof FGF19 and/or FGFR4

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS 1NMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

Definitions

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 ug/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25 C with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. Mol.Biol 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term “FGF19” (interchangeably termed “Fibroblast growth factor 19”),as used herein, refers, unless specifically or contextually indicatedotherwise, to any native or variant (whether native or synthetic) FGF19polypeptide. The term “native sequence” specifically encompassesnaturally occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants. The term “wildtype FGF19” generally refers to a polypeptide comprising the amino acidsequence of a naturally occurring FGF19 protein. The term “wild typeFGF19 sequence” generally refers to an amino acid sequence found in anaturally occurring FGF19.

The term “FGFR4” (interchangeably termed “Fibroblast growth factorreceptor 4”), as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) FGFR4 polypeptide. The term “native sequence”specifically encompasses naturally occurring truncated or secreted forms(e.g., an extracellular domain sequence), naturally occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants. The term “wild type FGFR4” generally refers to apolypeptide comprising the amino acid sequence of a naturally occurringFGFR4 protein. The term “wild type FGFR4 sequence” generally refers toan amino acid sequence found in a naturally occurring FGFR4.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CHI) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In one embodiment, an antibody fragment comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcRn binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For e.g., such an antibodyfragment may comprise on antigen binding arm linked to an Fc sequencecapable of conferring in vivo stability to the fragment.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures.

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 (L1), 46-56 (L2) and 89-97 (L3) in the VL and 26-35 (H1),49-65 or 50 to 65 (H2) and 93-102 (H3) in the VH. The variable domainresidues are numbered according to Kabat et al., supra for each of thesedefinitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: Clq bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or Presta U.S. Pat. No. 6,737,056 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins.

WO00/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. The content of that patent publication isspecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass)which are bound to their cognate antigen: To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased Clq binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

A “biological sample” (interchangeably termed “sample” or “tissue orcell sample”) encompasses a variety of sample types obtained from anindividual and can be used in a diagnostic or monitoring assay. Thedefinition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The sourceof the biological sample may be solid tissue as from a fresh, frozenand/or preserved organ or tissue sample or biopsy or aspirate; blood orany blood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the subject. In some embodiments,the biological sample is obtained from a primary or metastatic tumor.The biological sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention. In some embodiments, the same section of tissuesample is analyzed at both morphological and molecular levels, or isanalyzed with respect to both protein and nucleic acid.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

A “medicament” is an active drug to treat the disorder in question orits symptoms, or side effects.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; carcinoma, blastoma, and sarcoma.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, pituitary cancer, esophageal cancer,astrocytoma, soft tissue sarcoma, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer,testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastriccancer, melanoma, and various types of head and neck cancer.Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both non-neoplastic and neoplastic conditions. Neoplasticsinclude but are not limited those described above. Non-neoplasticdisorders include but are not limited to undesired or aberranthypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabeticand other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

The term “wasting” disorders (e.g., wasting syndrome, cachexia,sarcopenia) refers to a disorder caused by undesirable and/or unhealthyloss of weight or loss of body cell mass. In the elderly as well as inAIDS and cancer patients, wasting disease can result in undesired lossof body weight, including both the fat and the fat-free compartments.Wasting diseases can be the result of inadequate intake of food and/ormetabolic changes related to illness and/or the aging process. Cancerpatients and AIDS patients, as well as patients following extensivesurgery or having chronic infections, immunologic diseases,hyperthyroidism, Crohn's disease, psychogenic disease, chronic heartfailure or other severe trauma, frequently suffer from wasting diseasewhich is sometimes also referred to as cachexia, a metabolic and,sometimes, an eating disorder. Cachexia is additionally characterized byhypermetabolism and hypercatabolism. Although cachexia and wastingdisease are frequently used interchangeably to refer to wastingconditions, there is at least one body of research which differentiatescachexia from wasting syndrome as a loss of fat-free mass, andparticularly, body cell mass (Mayer, 1999, J. Nutr. 129 (ISSuppl.):256S-259S). Sarcopenia, yet another such disorder which canaffect the aging individual, is typically characterized by loss ofmuscle mass. End stage wasting disease as described above can develop inindividuals suffering from either cachexia or sarcopenia.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Forexample, an anti-angiogenesis agent is an antibody or other antagonistto an angiogenic agent as defined above, e.g., antibodies to VEGF,antibodies to VEGF receptors, small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinibmalate), AMG706). Anti-angiogenesis agents also include nativeangiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g.,Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit andDetmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing antiangiogenic factors); and,Sato Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 listsAnti-angiogenic agents used in clinical trials).

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows), sport animals, pets (such as cats, dogs and horses), primates,mice and rats.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammaII and calicheamicin omegaII (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin; azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, RatH-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingFGF19) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing FGF19) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-Iyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the polypeptide or by recombinant engineering thenucleic acid encoding the polypeptide. Accordingly, a compositioncomprising a polypeptide having an Fc region according to this inventioncan comprise polypeptides with K447, with all K447 removed, or a mixtureof polypeptides with and without the K447 residue.

Compositions of the Invention and Methods of Making Same

This invention encompasses compositions, including pharmaceuticalcompositions, comprising an anti-FGF19 antibody; and polynucleotidescomprising sequences encoding an anti-FGF19 antibody. As used herein,compositions comprise one or more antibodies that bind to FGF19, and/orone or more polynucleotides comprising sequences encoding one or moreantibodies that bind to FGF19. These compositions may further comprisesuitable carriers, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

The invention also encompasses isolated antibody and polynucleotideembodiments. The invention also encompasses substantially pure antibodyand polynucleotide embodiments.

The anti-FGF19 antibodies of the invention are preferably monoclonal.Also encompassed within the scope of the invention are Fab, Fab′,Fab′-SH and F(ab′)₂ fragments of the anti-FGF19 antibodies providedherein. These antibody fragments can be created by traditional means,such as enzymatic digestion, or may be generated by recombinanttechniques. Such antibody fragments may be chimeric or humanized. Thesefragments are useful for the diagnostic and therapeutic purposes setforth below.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-FGF19 monoclonal antibodies of the invention can be made usingthe hybridoma method first described by Kohler et al, Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to FGF19 generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of FGF19 and an adjuvant. FGF19 may be prepared using methodswell-known in the art, some of which are further described herein. Forexample, recombinant production of FGF19 is described below. In oneembodiment, animals are immunized with a derivative of FGF19 thatcontains the extracellular domain (ECD) of FGF19 fused to the Fc portionof an immunoglobulin heavy chain. In one embodiment, animals areimmunized with an FGF19-IgG1 fusion protein. Animals ordinarily areimmunized against immunogenic conjugates or derivatives of FGF19 withmonophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (RibiImmunochem. Research, Inc., Hamilton, Mont.) and the solution isinjected intradermally at multiple sites. Two weeks later the animalsare boosted. 7 to 14 days later animals are bled and the serum isassayed for anti-FGF19 titer. Animals are boosted until titer plateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against FGF19. Preferably,the binding specificity of monoclonal antibodies produced by hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbentassay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The anti-FGF19 antibodies of the invention can be made by usingcombinatorial libraries to screen for synthetic antibody clones with thedesired activity or activities. In principle, synthetic antibody clonesare selected by screening phage libraries containing phage that displayvarious fragments of antibody variable region (Fv) fused to phage coatprotein. Such phage libraries are panned by affinity chromatographyagainst the desired antigen. Clones expressing Fv fragments capable ofbinding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clonesare then eluted from the antigen, and can be further enriched byadditional cycles of antigen adsorption/elution. Any of the anti-FGF19antibodies of the invention can be obtained by designing a suitableantigen screening procedure to select for the phage clone of interestfollowed by construction of a full length anti-FGF19 antibody cloneusing the Fv sequences from the phage clone of interest and suitableconstant region (Fc) sequences described in Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-FGF19 clones is desired, the subject is immunized withFGF19 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-human FGF19clones is obtained by generating an anti-human FGF19 antibody responsein transgenic mice carrying a functional human immunoglobulin gene array(and lacking a functional endogenous antibody production system) suchthat FGF19 immunization gives rise to B cells producing human antibodiesagainst FGF19. The generation of human antibody-producing transgenicmice is described below.

Additional enrichment for anti-FGF19 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing FGF19-specific membrane bound antibody, e.g., by cellseparation with FGF19 affinity chromatography or adsorption of cells tofluorochrome-labeled FGF19 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which FGF19 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al, Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991). Repertoires ofsynthetically rearranged V genes can be derived in vitro from V genesegments. Most of the human VH-gene segments have been cloned andsequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-798(1992)), and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94(1993); these cloned segments (including all the major conformations ofthe H1 and H2 loop) can be used to generate diverse VH gene repertoireswith PCR primers encoding H3 loops of diverse sequence and length asdescribed in Hoogenboom and Winter, J. Mol. 227: 381-388 (1992). VHrepertoires can also be made with all the sequence diversity focused ina long H3 loop of a single length as described in Barbas et al., Proc.Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human Vκ and Vλ segmentshave been cloned and sequenced (reported in Williams and Winter, Eur. J.Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic lightchain repertoires. Synthetic V gene repertoires, based on a range of VHand VL folds, and L3 and H3 lengths, will encode antibodies ofconsiderable structural diversity. Following amplification of V-geneencoding DNAs, germline V-gene segments can be rearranged in vitroaccording to the methods of Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al, Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombinationapproach exploits the two-chain nature of Fab fragments to overcome thelimit on library size imposed by E. coli transformation efficiency.Naive VH and VL repertoires are cloned separately, one into a phagemidand the other into a phage vector. The two libraries are then combinedby phage infection of phagemid-containing bacteria so that each cellcontains a different combination and the library size is limited only bythe number of cells present (about 10¹² clones). Both vectors contain invivo recombination signals so that the VH and VL genes are recombinedonto a single replicon and are co-packaged into phage virions. Thesehuge libraries provide large numbers of diverse antibodies of goodaffinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹M range.

Nucleic acid sequence encoding an FGF19 can be designed using the aminoacid sequence of the desired region of FGF19. Alternatively, the cDNAsequence (or fragments thereof) may be used. Additional FGF19 sequencesare further disclosed in, e.g., NM_(—)022963, and Xie et al. (1999)Cytokine 11:729-735. DNAs encoding FGF19 can be prepared by a variety ofmethods known in the art. These methods include, but are not limited to,chemical synthesis by any of the methods described in Engels et al.,Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as the triester,phosphite, phosphoramidite and H-phosphonate methods. In one embodiment,codons preferred by the expression host cell are used in the design ofthe FGF19-encoding DNA. Alternatively, DNA encoding FGF19 can beisolated from a genomic or cDNA library.

Following construction of the DNA molecule encoding FGF19, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. In general,plasmid vectors contain replication and control sequences which arederived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells. Suitable vectors for expression in prokaryotic andeukaryotic host cells are known in the art and some are furtherdescribed herein. Eukaryotic organisms, such as yeasts, or cells derivedfrom multicellular organisms, such as mammals, may be used.

Optionally, the DNA encoding FGF19 is operably linked to a secretoryleader sequence resulting in secretion of the expression product by thehost cell into the culture medium. Examples of secretory leadersequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephosphatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Methods fortransfection are well known in the art, and some are further describedherein.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Methods fortransformation are well known in the art, and some are further describedherein.

Prokaryotic host cells used to produce FGF19 can be cultured asdescribed generally in Sambrook et al., supra.

The mammalian host cells used to produce FGF19 can be cultured in avariety of media, which is well known in the art and some of which isdescribed herein.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Purification of FGF19 may be accomplished using art-recognized methods.

The purified FGF19 can be attached to a suitable matrix such as agarosebeads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the FGF19 protein to the matrix can be accomplished by themethods described in Methods in Enzymology, vol. 44 (1976). A commonlyemployed technique for attaching protein ligands to polysaccharidematrices, e.g. agarose, dextran or cellulose, involves activation of thecarrier with cyanogen halides and subsequent coupling of the peptideligand's primary aliphatic or aromatic amines to the activated matrix.

Alternatively, FGF19 can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other art-known method for panning phage displaylibraries.

The phage library samples are contacted with immobilized FGF19 underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by FGF19 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for FGF19.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting FGF19, rare high affinity phage could becompeted out. To retain all the higher affinity mutants, phages can beincubated with excess biotinylated FGF19, but with the biotinylatedFGF19 at a concentration of lower molarity than the target molaraffinity constant for FGF19. The high affinity-binding phages can thenbe captured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics. Anti-FGF19 clones may also be activity selected.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones of the invention is readily isolated and sequencedusing conventional procedures (e.g. by using oligonucleotide primersdesigned to specifically amplify the heavy and light chain codingregions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding anti-FGF19 antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Human Antibodies

Human anti-FGF19 antibodies of the invention can be constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalanti-FGF19 antibodies of the invention can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forFGF19 and the other is for any other antigen. Exemplary bispecificantibodies may bind to two different epitopes of the FGF19 protein.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express FGF19. These antibodies possess an FGF19-binding armand an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH₂, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human. T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et at, Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fe region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc,wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. Such altering includes deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: asp, glu;    -   (4) basic: his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine. In accordance with this description and the teachings ofthe art, it is contemplated that in some embodiments, an antibody usedin methods of the invention may comprise one or more alterations ascompared to the wild type counterpart antibody, e.g. in the Fc region.These antibodies would nonetheless retain substantially the samecharacteristics required for therapeutic utility as compared to theirwild type counterpart. For example, it is thought that certainalterations can be made in the Fc region that would result in altered(i.e., either improved or diminished) Clq binding and/or ComplementDependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See alsoDuncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S.Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fcregion variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman)describe antibody variants with improved or diminished binding to FcRs.The content of these patent publications are specifically incorporatedherein by reference. See, also, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half lives and improvedbinding to the neonatal Fc receptor (FcRn), which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are describedin US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc regon with one or more substitutions therein which improve binding of theFc region to FcRn. Polypeptide variants with altered Fc region aminoacid sequences and increased or decreased Clq binding capability aredescribed in U.S. Pat. No. 6,194,551B1, WO99/51642. The contents ofthose patent publications are specifically incorporated herein byreference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Screening for Antibodies with Desired Properties

The antibodies of the invention bind FGF19, and in some embodiments, maymodulate one or more aspects of FGF19-associated effects, including butnot limited to FGFR4 activation, FGFR4 downstream molecular signaling,disruption of FGFR4 binding to FGF19, FGFR4 multimerization, expressionof a CYP7α1 gene, phosphorylation of FGFR4, MAPK, FRS2 and/or ERK2,activation of β-catenin, FGF19-promoted cell migration, and/ordisruption of any biologically relevant FGF19 and/or FGFR4 biologicalpathway, and/or treatment and/or prevention of a tumor, cellproliferative disorder or a cancer; and/or treatment or prevention of adisorder associated with FGF19 expression and/or activity (such asincreased FGF19 expression and/or activity).

The purified antibodies can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. Illustrative antigen binding assay are providedbelow in the Examples section.

In some embodiments, the invention provides anti-FGF19 monoclonalantibodies that compete with an antibody comprising a light chainvariable domain having the sequence:

DIKMTQSPSSMYASLGERVTIPCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKVEIKR (SEQ ID NO:4) and a heavy chainvariable domain having the sequence:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLITYGVHWVRQSPGKGLEWLGVIWPGGGTDYNAAFISRLSITKDNSKSQVFFKMNSLLANDTAIYFCVRKEYANLYAMDYWGQGTLLTVSA (SEQ ID NO:8) forbinding to FGF19. Such competitor antibodies include antibodies thatrecognize an FGF19 epitope that is the same as or overlaps with theFGF19 epitope recognized by the antibody. Such competitor antibodies canbe obtained by screening anti-FGF19 hybridoma supernatants for bindingto immobilized FGF19 in competition with labeled antibody comprising alight chain variable domain having the sequence:DIKMTQSPSSMYASLGERVTIPCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKVEIKR (SEQ ID NO:4) and a heavy chainvariable domain having the sequence:QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWPGGGTDYNAAFISRLSITKDNSKSQVFFKMNSLLANDTAIYFCVRKEYANLYAMDYWGQGTLLTVSA (SEQ ID NO:8). Ahybridoma supernatant containing competitor antibody will reduce theamount of bound, labeled antibody detected in the subject competitionbinding mixture as compared to the amount of bound, labeled antibodydetected in a control binding mixture containing irrelevant (or no)antibody. Any of the competition binding assays described herein aresuitable for use in the foregoing procedure.

Anti-FGF19 antibodies of the invention possessing the propertiesdescribed herein can be obtained by screening anti-FGF19 hybridomaclones for the desired properties by any convenient method. For example,if an anti-FGF19 monoclonal antibody that blocks or does not block thebinding of FGFR4 to FGF19 is desired, the candidate antibody can betested in a binding competition assay. Competition assays are well knownin the art, and one such assay is described in the Examples.

Other functional assays to determine the inhibitory capacity ofanti-FGF19 antibodies are known in the art, some of which areexemplified herein.

In some embodiments, the present invention contemplates alteredantibodies that possess some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fe activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fereceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). Clq binding assays may also be carried outto confirm that the antibody is unable to bind Clq and hence lacks CDCactivity. To assess complement activation, a CDC assay, e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art.

In some embodiments, the invention provides altered antibodies thatpossess increased effector functions and/or increased half-life.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41karat (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coliλ 1776 (ATCC31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examplesare illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component Expression and cloning vectors maycontain a selection gene, also termed a selectable marker. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, whererelevant, or (c) supply critical nutrients not available from complexmedia.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively; host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the anti-FGF19antibodies described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (eg., above). Enzymatically active toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁^(I) (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAN, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

v. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either galactose oxidase or sodium meta-periodate mayyield carbonyl (aldehyde and ketone) groups in the protein that canreact with appropriate groups on the drug (Hermanson, BioconjugateTechniques). In another embodiment, proteins containing N-terminalserine or threonine residues can react with sodium meta-periodate,resulting in production of an aldehyde in place of the first amino acid(Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No.5,362,852). Such aldehyde can be reacted with a drug moiety or linkernucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the present invention may be used in, for example, invitro, ex vivo and in vivo therapeutic methods.

The invention provides methods and compositions useful for modulatingdisease states associated with expression and/or activity of FGF19and/or FGFR4, such as increased expression and/or activity or undesiredexpression and/or activity, said methods comprising administration of aneffective dose of an anti-FGF19 antibody to an individual in need ofsuch treatment. In some embodiments, the disease state is associatedwith increased expression of FGF19, and the disease state comprisescholestasis or dysregulation of bile acid metabolism.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder, the methodscomprising administering an effective amount of an anti-FGF19 antibodyto an individual in need of such treatment.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder associated withincreased expression and/or activity of FGF19, the methods comprisingadministering an effective amount of an anti-FGF19 antibody to anindividual in need of such treatment.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder associated withincreased expression and/or activity of FGFR4, the methods comprisingadministering an effective amount of an anti-FGF19 antibody to anindividual in need of such treatment.

In one aspect, the invention provides methods for treating and/orpreventing a liver disorder, the methods comprising administering aneffective amount of an anti-FGF19 antibody to an individual in need ofsuch treatment In some embodiments, the liver disorder is cirrhosis.

In one aspect, the invention provides methods for treating and/orpreventing a wasting disorder, the methods comprising administering aneffective amount of an anti-FGF19 antibody to an individual in need ofsuch treatment. In some embodiments, the individual has a tumor, acancer, and/or a cell proliferative disorder.

It is understood that any suitable anti-FGF19 antibody may be used inmethods of treatment, including monoclonal and/or polyclonal antibodies,a human antibody, a chimeric antibody, an affinity-matured antibody, ahumanized antibody, and/or an antibody fragment. In some embodiments,any anti-FGF19 antibody described herein is used for treatment.

Moreover, at least some of the antibodies of the invention can bindantigen from other species. Accordingly, the antibodies of the inventioncan be used to bind specific antigen activity, e.g., in a cell culturecontaining the antigen, in human subjects or in other mammalian subjectshaving the antigen with which an antibody of the invention cross-reacts(e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig ormouse). In one embodiment, the antibody of the invention can be used forinhibiting antigen activities by contacting the antibody with theantigen such that antigen activity is inhibited. Preferably, the antigenis a human protein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor binding an antigen in an individual suffering from a disorderassociated with increased antigen expression and/or activity, comprisingadministering to the subject an antibody of the invention such that theantigen in the subject is bound. Preferably, the antigen is a humanprotein molecule and the subject is a human subject. Alternatively, thesubject can be a mammal expressing the antigen with which an antibody ofthe invention binds. Still further the subject can be a mammal intowhich the antigen has been introduced (e.g., by administration of theantigen or by expression of an antigen transgene). An antibody of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, an antibody of the invention can be administered toa non-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration).

The antibodies of the invention can be used to treat, inhibit, delayprogression of, prevent/delay recurrence of, ameliorate, or preventdiseases, disorders or conditions associated with expression and/oractivity of one or more antigen molecules.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with one or more cytotoxic agent(s) is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell. In oneembodiment, the cytotoxic agent targets or interferes with microtubulepolymerization. Examples of such cytotoxic agents include any of thechemotherapeutic agents noted herein (such as a maytansinoid,auristatin, dolastatin, or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

In any of the methods herein, one may administer to the subject orpatient along with the antibody herein an effective amount of a secondmedicament (where the antibody herein is a first medicament), which isanother active agent that can treat the condition in the subject thatrequires treatment. For instance, an antibody of the invention may beco-administered with another antibody, chemotherapeutic agent(s)(including cocktails of chemotherapeutic agents), anti-angiogenicagent(s), immunosuppressive agents(s), cytokine(s), cytokineantagonist(s), and/or growth-inhibitory agent(s). The type of suchsecond medicament depends on various factors, including the type ofdisorder, such as cancer or an autoimmune disorder, the severity of thedisease, the condition and age of the patient, the type and dose offirst medicament employed, etc.

Where an antibody of the invention inhibits tumor growth, for example,it may be particularly desirable to combine it with one or more othertherapeutic agents that also inhibit tumor growth. For instance, anantibody of the invention may be combined with an anti-angiogenic agent,such as an anti-VEGF antibody (e.g., AVASTIN®) and/or anti-ErbBantibodies (e.g. HERCEPTIN® trastuzumab anti-HER2 antibody or ananti-HER2 antibody that binds to Domain II of HER2, such as OMNITARG™pertuzumab anti-HER2 antibody) in a treatment scheme, e.g. in treatingany of the disease described herein, including colorectal cancer, lungcancer, hepatocellular carcinoma, breast cancer and/or pancreaticcancer. Alternatively, or additionally, the patient may receive combinedradiation therapy (e.g. external beam irradiation or therapy with aradioactive labeled agent, such as an antibody). Such combined therapiesnoted above include combined administration (where the two or moreagents are included in the same or separate formulations), and separateadministration, in which case, administration of the antibody of theinvention can occur prior to, and/or following, administration of theadjunct therapy or therapies. In addition, combining an antibody of thisinvention with a relatively non-cytotoxic agent such as another biologicmolecule, e.g., another antibody is expected to reduce cytotoxicityversus combining the antibody with a chemotherapeutic agent of otheragent that is highly toxic to cells.

Treatment with a combination of the antibody herein with one or moresecond medicaments preferably results in an improvement in the signs orsymptoms of cancer. For instance, such therapy may result in animprovement in survival (overall survival and/or progression-freesurvival) relative to a patient treated with the second medicament only(e.g., a chemotherapeutic agent only), and/or may result in an objectiveresponse *(partial or complete, preferably complete). Moreover,treatment with the combination of an antibody herein and one or moresecond medicament(s) preferably results in an additive, and morepreferably synergistic (or greater than additive), therapeutic benefitto the patient. Preferably, in this combination method the timingbetween at least one administration of the second medicament and atleast one administration of the antibody herein is about one month orless, more preferably, about two weeks or less.

For treatment of cancers, the second medicament is preferably anotherantibody, chemotherapeutic agent (including cocktails ofchemotherapeutic agents), anti-angiogenic agent, immunosuppressiveagent, prodrug, cytokine, cytokine antagonist, cytotoxic radiotherapy,corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascularagent, and/or growth-inhibitory agent. The cytotoxic agent includes anagent interacting with DNA, the antimetabolites, the topoisomerase I orII inhibitors, or the spindle inhibitor or stabilizer agents (e.g.,preferably vinca alkaloid, more preferably selected from vinblastine,deoxyvinblastine, vincristine, vindesine, vinorelbine, vinepidine,vinfosiltine, vinzolidine and vinfunine), or any agent used inchemotherapy such as 5-FU, a taxane, doxorubicin, or dexamethasone.

In another embodiment, the second medicament is another antibody used totreat cancers such as those directed against the extracellular domain ofthe HER2/neu receptor, e.g., trastuzumab, or one of its functionalfragments, pan-HER inhibitor, a Src inhibitor, a MEK inhibitor, or anEGFR inhibitor (e.g., an anti-EGFR antibody (such as one inhibiting thetyrosine kinase activity of the EGFR), which is preferably the mousemonoclonal antibody 225, its mouse-man chimeric derivative C225, or ahumanized antibody derived from this antibody 225 or derived naturalagents, dianilinophthalimides, pyrazolo- or pyrrolopyridopyrimidines,quinazilines, gefitinib, erlotinib, cetuximab, ABX-EFG, canertinib,EKB-569 and PKI-166), or dual-EGFR/HER-2 inhibitor such as lapatanib.Additional second medicaments include alemtuzumab (CAMPATH™), FavID(IDKLH), CD20 antibodies with altered glycosylation, such asGA-101/GLYCART™, oblimersen (GENASENSE™), thalidomide and analogsthereof, such as lenalidomide (REVLIMID™), imatinib, sorafenib,ofatumumab (HUMAX-CD20™), anti-CD40 antibody, e.g. SGN-40, andanti-CD-80 antibody, e.g. galiximab:

The anti-emetic agent is preferably ondansetron hydrochloride,granisetron hydrochloride, metroclopramide, domperidone, haloperidol,cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine,or tropisetron. The vaccine is preferably GM-CSF DNA and cell-basedvaccines, dendritic cell vaccine, recombinant viral vaccines, heat shockprotein (HSP) vaccines, allogeneic or autologous tumor vaccines. Theanalgesic agent preferably is ibuprofen, naproxen, choline magnesiumtrisalicylate, or oxycodone hydrochloride. The anti-vascular agentpreferably is bevacizumab, or rhuMAb-VEGF. Further second medicamentsinclude anti-proliferative agents such a farnesyl protein transferaseinhibitors, anti-VEGF inhibitors, p53 inhibitors, or PDGFR inhibitors.The second medicament herein includes also biologic-targeted therapysuch as treatment with antibodies as well as small-molecule-targetedtherapy, for example, against certain receptors.

Many anti-angiogenic agents have been identified and are known in theart, including those listed herein, e.g., listed under Definitions, andby, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al.,Nature Reviews: Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin.Oncol., 8:200-206 (2003). See also, US Patent Application US20030055006.In one embodiment, an anti-FGF19 antibody is used in combination with ananti-VEGF neutralizing antibody (or fragment) and/or another VEGFantagonist or a VEGF receptor antagonist including, but not limited to,for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3,neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of blockingVEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weightinhibitors of VEGFR tyrosine kinases (RTK), antisense strategies forVEGF, ribozymes against VEGF or VEGF receptors, antagonist variants ofVEGF; and any combinations thereof. Alternatively, or additionally, twoor more angiogenesis inhibitors may optionally be co-administered to thepatient in addition to VEGF antagonist and other agent. In certainembodiment, one or more additional therapeutic agents, e.g., anti-canceragents, can be administered in combination with anti-FGF19 antibody, theVEGF antagonist, and an anti-angiogenesis agent.

Chemotherapeutic agents useful herein are described supra, e.g., in thedefinition of “chemotherapeutic agent”.

Exemplary second medicaments include an alkylating agent, a folateantagonist, a pyrimidine antagonist, a cytotoxic antibiotic, a platinumcompound or platinum-based compound, a taxane, a vinca alkaloid, a c-Kitinhibitor, a topoisomerase inhibitor, an anti-angiogenesis inhibitorsuch as an anti-VEGF inhibitor, a HER-2 inhibitor, an EGFR inhibitor ordual EGFR/HER-2 kinase inhibitor, an anti-estrogen such as fulvestrant,and a hormonal therapy agent, such as carboplatin, cisplatin,gemcitabine, capecitabine, epirubicin, tamoxifen, an aromataseinhibitor, and prednisone. Most preferably, the cancer is colorectalcancer and the second medicament is an EGFR inhibitor such as erlotinib,an anti-VEGF inhibitor such as bevacizumab, or is cetuximab, arinotecan,irinotecan, or FOLFOX, or the cancer is breast cancer an the secondmedicament is an anti-estrogen modulator such as fulvestrant, tamoxifenor an aromatase inhibitor such as letrozole, exemestane, or anastrozole,or is a VEGF inhibitor such as bevacizumab, or is a chemotherapeuticagent such as doxorubicin, and/or a taxane such as paclitaxel, or is ananti-HER-2 inhibitor such as trastuzumab, or a dual EGFR/HER-2 kinaseinhibitor such as lapatinib or a HER-2 downregulator such as 17AAG(geldanamycin derivative that is a heat shock protein [Hsp] 90 poison)(for example, for breast cancers that have progressed on trastuzumab).In other embodiments, the cancer is lung cancer, such as small-cell lungcancer, and the second medicament is a VEGF inhibitor such asbevacizumab, or an EGFR inhibitor such as, e.g., erlotinib or a c-Kitinhibitor such as e.g., imatinib. In other embodiments, the cancer isliver cancer, such as hepatocellular carcinoma, and the secondmedicament is an EGFR inhibitor such as erlotinib, a chemotherapeuticagent such as doxorubicin or irinotecan, a taxane such as paclitaxel,thalidomide and/or interferon. Further, a preferred chemotherapeuticagent for front-line therapy of cancer is taxotere, alone in combinationwith other second medicaments. Most preferably, if chemotherapy isadministered, it is given first, followed by the antibodies herein.

Such second medicaments may be administered within 48 hours after theantibodies herein are administered, or within 24 hours, or within 12hours, or within 3-12 hours after said agent, or may be administeredover a pre-selected period of time, which is preferably about 1 to 2days. Further, the dose of such agent may be sub-therapeutic.

The antibodies herein can be administered concurrently, sequentially, oralternating with the second medicament or upon non-responsiveness withother therapy. Thus, the combined administration of a second medicamentincludes co-administration (concurrent administration), using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) medicaments simultaneously exert theirbiological activities. All these second medicaments may be used incombination with each other or by themselves with the first medicament,so that the express “second medicament” as used herein does not mean itis the only medicament besides the first medicament, respectively. Thus,the second medicament need not be one medicament, but may constitute orcomprise more than one such drug.

These second medicaments as set forth herein are generally used in thesame dosages and with administration routes as the first medicaments, orabout from 1 to 99% of the dosages of the first medicaments. If suchsecond medicaments are used at all, preferably, they are used in loweramounts than if the first medicament were not present, especially insubsequent dosings beyond the initial dosing with the first medicament,so as to eliminate or reduce side effects caused thereby.

The invention also provides methods and compositions for inhibiting orpreventing relapse tumor growth or relapse cancer cell growth. Relapsetumor growth or relapse cancer cell growth is used to describe acondition in which patients undergoing or treated with one or morecurrently available therapies (e.g., cancer therapies, such aschemotherapy, radiation therapy, surgery, hormonal therapy and/orbiological therapy/immunotherapy, anti-VEGF antibody therapy,particularly a standard therapeutic regimen for the particular cancer)is not clinically adequate to treat the patients or the patients are nolonger receiving any beneficial effect from the therapy such that thesepatients need additional effective therapy. As used herein, the phrasecan also refer to a condition of the “non-responsive/refractory”patient, e.g., which describe patients who respond to therapy yet sufferfrom side effects, develop resistance, do not respond to the therapy, donot respond satisfactorily to the therapy, etc. In various embodiments,a cancer is relapse tumor growth or relapse cancer cell growth where thenumber of cancer cells has not been significantly reduced, or hasincreased, or tumor size has not been significantly reduced, or hasincreased, or fails any further reduction in size or in number of cancercells. The determination of whether the cancer cells are relapse tumorgrowth or relapse cancer cell growth can be made either in vivo or invitro by any method known in the art for assaying the effectiveness oftreatment on cancer cells, using the art-accepted meanings of “relapse”or “refractory” or “non-responsive” in such a context. A tumor resistantto anti-VEGF treatment is an example of a relapse tumor growth.

The invention provides methods of blocking or reducing relapse tumorgrowth or relapse cancer cell growth in a subject by administering oneor more anti-FGF19 antibody to block or reduce the relapse tumor growthor relapse cancer cell growth in subject. In certain embodiments, theantagonist can be administered subsequent to the cancer therapeutic. Incertain embodiments, the anti-FGF19 antibody is administeredsimultaneously with cancer therapy. Alternatively, or additionally, theanti-FGF19 antibody therapy alternates with another cancer therapy,which can be performed in any order. The invention also encompassesmethods for administering one or more inhibitory antibodies to preventthe onset or recurrence of cancer in patients predisposed to havingcancer. Generally, the subject was or is concurrently undergoing cancertherapy. In one embodiment, the cancer therapy is treatment with ananti-angiogenesis agent, e.g., a VEGF antagonist. The anti-angiogenesisagent includes those known in the art and those found under theDefinitions herein. In one embodiment, the anti-angiogenesis agent is ananti-VEGF neutralizing antibody or fragment (e.g., humanized A4.6.1,AVASTIN® (Genentech, South San Francisco, Calif.), Y0317, M4, G6, B20,2C3, etc.). See, e.g., U.S. Pat. Nos. 6,582,959, 6,884,879, 6,703,020;WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; US PatentApplications 20030206899, 20030190317, 20030203409, and 20050112126;Popkov et al., Journal of Immunological Methods 288:149-164 (2004); and,WO2005012359. Additional agents can be administered in combination withVEGF antagonist and an anti-FGF19 antibody for blocking or reducingrelapse tumor growth or relapse cancer cell growth.

The antibodies of the invention (and adjunct therapeutic agent) is/areadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibodies are suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, e.g. by injections, suchas intravenous or subcutaneous injections, depending in part on whetherthe administration is brief or chronic.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents will depend on the type of disease to be treated, the type ofantibody, the severity and course of the disease, whether the antibodyis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the antibody,and the discretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. One typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the antibody would be in the range from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, e.g. about six doses ofthe antibody). An initial higher loading dose, followed by one or morelower doses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

The anti-FGF19 antibodies of the invention are useful in assaysdetecting FGF19 expression (such as diagnostic or prognostic assays) inspecific cells or tissues wherein the antibodies are labeled asdescribed below and/or are immobilized on an insoluble matrix. However,it is understood that any suitable anti-FGF19 antibody may be used inembodiments involving detection and diagnosis. Some methods for makinganti-FGF19 antibodies are described herein and methods for makinganti-FGF19 antibodies are well known in the art.

In another aspect, the invention provides methods for detection ofFGF19, the methods comprising detecting FGF19-anti-FGF19 antibodycomplex in the sample. The term “detection” as used herein includesqualitative and/or quantitative detection (measuring levels) with orwithout reference to a control.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGF19 expression and/or activity, the methodscomprising detecting FGF19-anti-FGF19 antibody complex in a biologicalsample from an individual having or suspected of having the disorder. Insome embodiments, the FGF19 expression is increased expression orabnormal (undesired) expression.

In another aspect, the invention provides any of the anti-FGF19antibodies described herein, wherein the anti-FGF19 antibody comprises adetectable label.

In another aspect, the invention provides a complex of any of theanti-FGF19 antibodies described herein and FGF19. In some embodiments,the complex is in vivo or in vitro. In some embodiments, the complexcomprises a cancer cell. In some embodiments, the anti-FGF19 antibody isdetectably labeled.

Anti-FGF19 antibodies (e.g., any of the FGF19 antibodies describedherein) can be used for the detection of FGF19 in any one of a number ofwell known detection assay methods.

In one aspect, the invention provides methods for detecting a disorderassociated with FGF19 expression and/or activity, the methods comprisingdetecting FGF19 in a biological sample from an individual. In someembodiments, the FGF19 expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as colorectal cancer, lung cancer,hepatocellular carcinoma, breast cancer and/or pancreatic cancer. Insome embodiment, the biological sample is serum or of a tumor.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGF19expression in an individual's biological sample, if any; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGF19 expression detected instep (a). In some embodiments, increased FGF19 expression in theindividual's biological sample relative to a reference value or controlsample is detected. In some embodiments, decreased FGF19 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected in the individual. In some embodiments, FGF19expression is detected and treatment with an anti-FGF19 antibody isselected. Methods of treating a disorder with an anti-FGF19 antibody aredescribed herein and some methods are exemplified herein.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, furtherwherein FGF19 expression and/or FGFR4 is detected in cells and/or tissuefrom the human patient before, during or after administration of ananti-FGF19 antibody. In some embodiments, FGF19 over-expression isdetected before, during and/or after administration of an anti-FGF19antibody. In some embodiments, FGFR4 expression is detected before,during and/or after administration of an anti-FGF19 antibody. Expressionmay be detected before; during; after; before and during; before andafter, during and after; or before, during and after administration ofan anti-FGF19 antibody. Methods of treating a disorder with ananti-FGF19 antibody are described herein and some methods areexemplified herein.

For example, a biological sample may be assayed for FGF19 by obtainingthe sample from a desired source, admixing the sample with anti-FGF19antibody to allow the antibody to form antibody/FGF19 complex with anyFGF19 present in the mixture, and detecting any antibody/FGF19 complexpresent in the mixture. The biological sample may be prepared for assayby methods known in the art which are suitable for the particularsample. The methods of admixing the sample with antibodies and themethods of detecting antibody/FGF19 complex are chosen according to thetype of assay used. Such assays include immunohistochemistry,competitive and sandwich assays, and steric inhibition assays. Forsample preparation, a tissue or cell sample from a mammal (typically ahuman patient) may be used. Examples of samples include, but are notlimited to, cancer cells such as colon, breast, prostate, ovary, lung,stomach, pancreas, lymphoma, and leukemia cancer cells. FGF19 may alsobe measured in serum. The sample can be obtained by a variety ofprocedures known in the art including, but not limited to surgicalexcision, aspiration or biopsy. The tissue may be fresh or frozen. Inone embodiment, the sample is fixed and embedded in paraffin or thelike. The tissue sample may be fixed (i.e. preserved) by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology,” 3^(rd) edition (1960) Lee G. Luna,H T (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, NewYork; The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, ArmedForces Institute of Pathology, American Registry of Pathology,Washington, D.C.). One of ordinary skill in the art will appreciate thatthe choice of a fixative is determined by the purpose for which thesample is to be histologically stained or otherwise analyzed. One ofordinary skill in the art will also appreciate that the length offixation depends upon the size of the tissue sample and the fixativeused. By way of example, neutral buffered formalin, Bouin's orparaformaldehyde, may be used to fix a sample. Generally, the sample isfirst fixed and is then dehydrated through an ascending series ofalcohols, infiltrated and embedded with paraffin or other sectioningmedia so that the tissue sample may be sectioned. Alternatively, one maysection the tissue and fix the sections obtained. By way of example, thetissue sample may be embedded and processed in paraffin by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology”, supra). Examples of paraffin thatmay be used include, but are not limited to, Paraplast, Broloid, andTissuemay. Once the tissue sample is embedded, the sample may besectioned by a microtome or the like (See e.g., “Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology”, supra). Byway of example for this procedure, sections may range from about threemicrons to about five microns in thickness. Once sectioned, the sectionsmay be attached to slides by several standard methods. Examples of slideadhesives include, but are not limited to, silane, gelatin,poly-L-lysine and the like. By way of example, the paraffin embeddedsections may be attached to positively charged slides and/or slidescoated with poly-L-lysine. If paraffin has been used as the embeddingmaterial, the tissue sections are generally deparaffinized andrehydrated to water. The tissue sections may be deparaffinized byseveral conventional standard methodologies. For example, xylenes and agradually descending series of alcohols may be used (See e.g., “Manualof Histological Staining Method of the Armed Forces Institute ofPathology”, supra). Alternatively, commercially availabledeparaffinizing non-organic agents such as Hemo-De7 (CMS, Houston, Tex.)may be used.

Analytical methods for FGF19 all use one or more of the followingreagents: labeled FGF19 analogue, immobilized FGF19 analogue, labeledanti-FGF19 antibody, immobilized anti-FGF19 antibody and stericconjugates. The labeled reagents also are known as “tracers.”

The label used is any detectable functionality that does not interferewith the binding of FGF19 and anti-FGF19 antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected.

The label used is any detectable functionality that does not interferewith the binding of FGF19 and anti-FGF19 antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412(1982). Preferred labels herein are enzymes such as horseradishperoxidase and alkaline phosphatase. The conjugation of such label,including the enzymes, to the antibody is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the anti-FGF19 antibody from any FGF19that remains free in solution. This conventionally is accomplished byeither insolubilizing the anti-FGF19 antibody or FGF19 analogue beforethe assay procedure, as by adsorption to a water-insoluble matrix orsurface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling(for example, using glutaraldehyde cross-linking), or by insolubilizingthe anti-FGF19 antibody or FGF19 analogue afterward, e.g., byimmunoprecipitation.

The expression of proteins in a sample may be examined usingimmunohistochemistry and staining protocols. Immunohistochemicalstaining of tissue sections has been shown to be a reliable method ofassessing or detecting presence of proteins in a sample.Immunohistochemistry (“IHC”) techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods. For sample preparation, a tissue or cell samplefrom a mammal (typically a human patient) may be used. The sample can beobtained by a variety of procedures known in the art including, but notlimited to surgical excision, aspiration or biopsy. The tissue may befresh or frozen. In one embodiment, the sample is fixed and embedded inparaffin or the like. The tissue sample may be fixed (i.e. preserved) byconventional methodology. One of ordinary skill in the art willappreciate that the choice of a fixative is determined by the purposefor which the sample is to be histologically stained or otherwiseanalyzed. One of ordinary skill in the art will also appreciate that thelength of fixation depends upon the size of the tissue sample and thefixative used.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigen(e.g., FGF19) is determined directly. This direct assay uses a labeledreagent, such as a fluorescent tag or an enzyme-labeled primaryantibody, which can be visualized without further antibody interaction.In a typical indirect assay, unconjugated primary antibody binds to theantigen and then a labeled secondary antibody binds to the primaryantibody. Where the secondary antibody is conjugated to an enzymaticlabel, a chromogenic or fluorogenic substrate is added to providevisualization of the antigen. Signal amplification occurs becauseseveral secondary antibodies may react with different epitopes on theprimary antibody.

The primary and/or secondary antibody used for immunohistochemistrytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired, For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out (see, e.g., Leong etal. Appl. Immunohistochem. 4(3):201 (1996)).

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation. The extent of binding ofantibody to the sample is determined by using any one of the detectablelabels discussed above. Preferably, the label is an enzymatic label(e.g. HRPO) which catalyzes a chemical alteration of the chromogenicsubstrate such as 3,3′-diaminobenzidine chromogen. Preferably theenzymatic label is conjugated to antibody which binds specifically tothe primary antibody (e.g. the primary antibody is rabbit polyclonalantibody and secondary antibody is goat anti-rabbit antibody).

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope, and stainingintensity criteria, routinely used in the art, may be employed.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer FGF19 analogue tocompete with the test sample FGF19 for a limited number of anti-FGF19antibody antigen-binding sites. The anti-FGF19 antibody generally isinsolubilized before or after the competition and then the tracer andFGF19 bound to the anti-FGF19 antibody are separated from the unboundtracer and FGF19. This separation is accomplished by decanting (wherethe binding partner was preinsolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample FGF19 is inversely proportional to the amount ofbound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of FGF19 are prepared andcompared with the test results to quantitatively determine the amount ofFGF19 present in the test sample. These assays are called ELISA systemswhen enzymes are used as the detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theFGF19 is prepared and used such that when anti-FGF19 antibody binds tothe FGF19 the presence of the anti-FGF19 antibody modifies the enzymeactivity. In this case, the FGF19 or its immunologically activefragments are conjugated with a bifunctional organic bridge to an enzymesuch as peroxidase. Conjugates are selected for use with anti-FGF19antibody so that binding of the anti-FGF19 antibody inhibits orpotentiates the enzyme activity of the label. This method per se iswidely practiced under the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small FGF19 fragment so that antibodyto hapten is substantially unable to bind the conjugate at the same timeas anti-FGF19 antibody. Under this assay procedure the FGF19 present inthe test sample will bind anti-FGF19 antibody, thereby allowinganti-hapten to bind the conjugate, resulting in a change in thecharacter of the conjugate hapten, e.g., a change in fluorescence whenthe hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of FGF19or anti-FGF19 antibodies. In sequential sandwich assays an immobilizedanti-FGF19 antibody is used to adsorb test sample FGF19, the test sampleis removed as by washing, the bound FGF19 is used to adsorb a second,labeled anti-FGF19 antibody and bound material is then separated fromresidual tracer. The amount of bound tracer is directly proportional totest sample FGF19. In “simultaneous” sandwich assays the test sample isnot separated before adding the labeled anti-FGF19. A sequentialsandwich assay using an anti-FGF19 monoclonal antibody as one antibodyand a polyclonal anti-FGF19 antibody as the other is useful in testingsamples for FGF19.

The foregoing are merely exemplary detection assays for FGF19. Othermethods now or hereafter developed that use anti-FGF19 antibody for thedetermination of FGF19 are included within the scope hereof, includingthe bioassays described herein.

In one aspect, the invention provides methods to detect (e.g., presenceor absence of or amount) a polynucleotide(s) (e.g., FGF19polynucleotides) in a biological sample from an individual, such as ahuman subject. A variety of methods for detecting polynucleotides can beemployed and include, for example, RT-PCR, TaqMan, amplificationmethods, polynucleotide microarray, and the like.

Methods for the detection of polynucleotides (such as mRNA) are wellknown and include, for example, hybridization assays using complementaryDNA probes (such as in situ hybridization using labeled FGF19riboprobes), Northern blot and related techniques, and various nucleicacid amplification assays (such as RT-PCR using complementary primersspecific for FGF19, and other amplification type detection methods, suchas, for example, branched DNA, SPIA, Ribo-SPIA, SISBA, TMA and thelike).

Biological samples from mammals can be conveniently assayed for, e.g.,FGF19 mRNAs using Northern, dot blot or PCR analysis. For example,RT-PCR assays such as quantitative PCR assays are well known in the art.In an illustrative embodiment of the invention, a method for detectingFGF19 mRNA in a biological sample comprises producing cDNA from thesample by reverse transcription using at least one primer; amplifyingthe cDNA so produced using an FGF19 polynucleotide as sense andantisense primers to amplify FGF19 cDNAs therein; and detecting thepresence or absence of the amplified FGF19 cDNA. In addition, suchmethods can include one or more steps that allow one to determine theamount (levels) of FGF19 mRNA in a biological sample (e.g. bysimultaneously examining the levels a comparative control mRNA sequenceof a housekeeping gene such as an actin family member). Optionally, thesequence of the amplified FGF19 cDNA can be determined.

Probes and/or primers may be labeled with a detectable marker, such as,for example, a radioisotope, fluorescent compound, bioluminescentcompound, a chemiluminescent compound, metal chelator or enzyme. Suchprobes and primers can be used to detect the presence of FGF19polynucleotides in a sample and as a means for detecting a cellexpressing FGF19 proteins. As will be understood by the skilled artisan,a great many different primers and probes may be prepared (e.g., basedon the sequences provided in herein) and used effectively to amplify,clone and/or determine the presence or absence of and/or amount of FGF19mRNAs.

Optional methods of the invention include protocols comprising detectionof polynucleotides, such as FGF19 polynucleotide, in a tissue or cellsample using microarray technologies. For example, using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. Theprobes are then hybridized to an array of nucleic acids immobilized on asolid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes that have potential to be expressed in certain disease statesmay be arrayed on a solid support. Hybridization of a labeled probe witha particular array member indicates that the sample from which the probewas derived expresses that gene. Differential gene expression analysisof disease tissue can provide valuable information. Microarraytechnology utilizes nucleic acid hybridization techniques and computingtechnology to evaluate the mRNA expression profile of thousands of geneswithin a single experiment. (see, e.g., WO 01/75166 published Oct. 11,2001; (See, for example, U.S. Pat. No. 5,700,637, U.S. Pat. No.5,445,934, and U.S. Pat. No. 5,807,522, Lockart, Nature Biotechnology,14:1675-1680 (1996); Cheung, V. G. et al., Nature Genetics21(Suppl):15-19 (1999) for a discussion of array fabrication). DNAmicroarrays are miniature arrays containing gene fragments that areeither synthesized directly onto or spotted onto glass or othersubstrates. Thousands of genes are usually represented in a singlearray. A typical microarray experiment involves the following steps: 1.preparation of fluorescently labeled target from RNA isolated from thesample, 2. hybridization of the labeled target to the microarray, 3.washing, staining, and scanning of the array, 4. analysis of the scannedimage and 5. generation of gene expression profiles. Currently two maintypes of DNA microarrays are being used: oligonucleotide (usually 25 to70 mers) arrays and gene expression arrays containing PCR productsprepared from cDNAs. In forming an array, oligonucleotides can be eitherprefabricated and spotted to the surface or directly synthesized on tothe surface (in situ).

The Affymetrix GeneChip® system is a commercially available microarraysystem which comprises arrays fabricated by direct synthesis ofoligonucleotides on a glass surface. Probe/Gene Arrays:Oligonucleotides, usually 25 mers, are directly synthesized onto a glasswafer by a combination of semiconductor-based photolithography and solidphase chemical synthesis technologies. Each array contains up to 400,000different oligos and each oligo is present in millions of copies. Sinceoligonucleotide probes are synthesized in known locations on the array,the hybridization patterns and signal intensities can be interpreted interms of gene identity and relative expression levels by the AffymetrixMicroarray Suite software. Each gene is represented on the array by aseries of different oligonucleotide probes. Each probe pair consists ofa perfect match oligonucleotide and a mismatch oligonucleotide. Theperfect match probe has a sequence exactly complimentary to theparticular gene and thus measures the expression of the gene. Themismatch probe differs from the perfect match probe by a single basesubstitution at the center base position, disturbing the binding of thetarget gene transcript. This helps to determine the background andnonspecific hybridization that contributes to the signal measured forthe perfect match oligo. The Microarray Suite software subtracts thehybridization intensities of the mismatch probes from those of theperfect match probes to determine the absolute or specific intensityvalue for each probe set. Probes are chosen based on current informationfrom GenBank and other nucleotide repositories. The sequences arebelieved to recognize unique regions of the 3′ end of the gene. AGeneChip Hybridization Oven (“rotisserie” oven) is used to carry out thehybridization of up to 64 arrays at one time. The fluidics stationperforms washing and staining of the probe arrays. It is completelyautomated and contains four modules, with each module holding one probearray. Each module is controlled independently through Microarray Suitesoftware using preprogrammed fluidics protocols. The scanner is aconfocal laser fluorescence scanner which measures fluorescenceintensity emitted by the labeled cRNA bound to the probe arrays. Thecomputer workstation with Microarray Suite software controls thefluidics station and the scanner. Microarray Suite software can controlup to eight fluidics stations using preprogrammed hybridization, wash,and stain protocols for the probe array. The software also acquires andconverts hybridization intensity data into a presence/absence call foreach gene using appropriate algorithms. Finally, the software detectschanges in gene expression between experiments by comparison analysisand formats the output into .txt files, which can be used with othersoftware programs for further data analysis.

In some embodiments, FGF19 gene deletion, gene mutation, or geneamplification is detected. Gene deletion, gene mutation, oramplification may be measured by any one of a wide variety of protocolsknown in the art, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis),or in situ hybridization (e.g., FISH), using an appropriately labeledprobe, cytogenetic methods or comparative genomic hybridization (CGH)using an appropriately labeled probe. In addition, these methods may beemployed to detect FGF19 ligand gene deletion, ligand mutation, or geneamplification. As used herein, “detecting FGF19 expression” encompassesdetection of FGF19 gene deletion, gene mutation or gene amplification.

Additionally, one can examine the methylation status of the FGF19 genein a tissue or cell sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells, and can result in alteredexpression of various genes. A variety of assays for examiningmethylation status of a gene are well known in the art. For example, onecan utilize, in Southern hybridization approaches, methylation-sensitiverestriction enzymes which cannot cleave sequences that containmethylated CpG sites to assess the methylation status of CpG islands. Inaddition, MSP (methylation specific PCR) can rapidly profile themethylation status of all the CpG sites present in a CpG island of agiven gene. This procedure involves initial modification of DNA bysodium bisulfite (which will convert all unmethylated cytosines touracil) followed by amplification using primers specific for methylatedversus unmethylated DNA. Protocols involving methylation interferencecan also be found for example in Current Protocols In Molecular Biology,Unit 12, Frederick M. Ausubel et al. eds., 1995; De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999); Brooks et al, Cancer Epidemiol.Biomarkers Prey., 1998, 7:531-536); and Lethe et al., Int. J. Cancer76(6): 903-908 (1998). As used herein, “detecting FGF19 expression”encompasses detection of FGF19 gene methylation.

The Examples of the present application disclose that FGFR4 is expressedin human primary liver, lung and colon tumors and in colon cancer celllines, and further that FGF19 and FGFR4 are co-expressed in humanprimary liver, lung and colon tumors and in colon cancer cell lines.Accordingly, in some embodiments, expression of FGFR4 polypeptide and/orpolynucleotide is detected (alone or in conjunction (simultaneouslyand/or sequentially)) with FGF19 expression) in a biological sample. Asdescribed above and in the art, it is presently believed that FGF19binds to the FGFR4 receptor. Using methods known in the art, includingthose described herein, the polynucleotide and/or polypeptide expressionof FGFR4 can be detected. By way of example, the IHC techniquesdescribed above may be employed to detect the presence of one of moresuch molecules in the sample. As used herein, “in conjunction” is meantto encompass any simultaneous and/or sequential detection. Thus, it iscontemplated that in embodiments in which a biological sample is beingexamined not only for the presence of FGF19, but also for the presenceof FGFR4, separate slides may be prepared from the same tissue orsample, and each slide tested with a reagent that binds to FGF19 and/orFGFR4, respectively. Alternatively, a single slide may be prepared fromthe tissue or cell sample, and antibodies directed to FGF19 and FGFR4may be used in connection with a multi-color staining protocol to allowvisualization and detection of the FGF19 and FGFR4.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGFR4 expression and/or activity, the methodscomprising detecting FGFR4 in a biological sample from an individual. Insome embodiments, FGFR4 expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as colorectal cancer, lung cancer,hepatocellular carcinoma, breast cancer and/or pancreatic cancer. Insome embodiment, the biological sample is serum or of a tumor.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGFR4 and FGF19 expression and/or activity, themethods comprising detecting FGFR4 and FGF19 in a biological sample froman individual. In some embodiments, the FGF19 expression is increasedexpression or abnormal expression. In some embodiments, FGFR4 expressionis increased expression or abnormal expression. In some embodiments, thedisorder is a tumor, cancer, and/or a cell proliferative disorder, suchas colorectal cancer, lung cancer, hepatocellular carcinoma, breastcancer and/or pancreatic cancer. In some embodiment, the biologicalsample is serum or of a tumor. In some embodiments, expression of FGFR4is detected in a first biological sample, and expression of FGF19 isdetected in a second biological sample.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGFR4expression in an individual's biological sample, if any; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGFR4 expression detected instep (a). In some embodiments, increased FGFR4 expression in theindividual's biological sample relative to a reference value or controlsample is detected. In some embodiments, decreased FGFR4 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected in the individual. In some embodiments, FGFR4expression is detected and treatment with an anti-FGF19 antibody isselected.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGF19and FGFR4 expression in the biological sample, if any; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGF19 and FGFR4 expressiondetected in step (a). In some embodiments, increased FGF19 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected. In some embodiments, decreased FGF19expression in the individual's biological sample relative to a referencevalue or control sample is detected in the individual. In someembodiments, increased FGFR4 expression in the individual's biologicalsample relative to a reference value or control sample is detected. Insome embodiments, decreased FGFR4 expression in the individual'sbiological sample relative to a reference value or control sample isdetected in the individual. In some embodiments, FGFR4 and FGF19expression are detected and treatment with an anti-FGF19 antibody isselected. In some embodiments, expression of FGFR4 is detected in afirst biological sample, and expression of FGF19 is detected in a secondbiological sample.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, furtherwherein FGF19 expression and/or FGFR4 is detected in cells and/or tissuefrom the human patient before, during or after administration of ananti-FGF19 antibody. In some embodiments, FGF19 over-expression isdetected before, during and/or after administration of an anti-FGF19antibody. In some embodiments, FGFR4 expression is detected before,during and/or after administration of an anti-FGF19 antibody. Expressionmay be detected before; during; after; before and during; before andafter; during and after; or before, during and after administration ofan anti-FGF19 antibody.

In some embodiments involving detection, expression of FGFR4 downstreammolecular signaling is detected in addition to or as an alternative todetection of FGFR4 detection. In some embodiments, detection of FGFR4downstream molecular signaling comprises one or more of detection ofphosphorylation of MAPK, FRS2 or ERK2.

Some embodiments involving detection further comprise detection of Wntpathway activation. In some embodiments, detection of Wnt pathwayactivation comprises one or more of tyrosine phosphorylation ofβ-catenin, expression of Wnt target genes, β-catenin mutation, andE-cadherin binding to β-catenin. Detection of Wnt pathway activation isknown in the art, and some examples are described and exemplifiedherein.

In some embodiments, the treatment is for a cancer selected from thegroup consisting of colorectal cancer, lung cancer, ovarian cancer,pituitary cancer, pancreatic cancer, mammary fibroadenoma, prostatecancer, head and neck squamous cell carcinoma, soft tissue sarcoma,breast cancer, neuroblastomas, melanoma, breast carcinoma, gastriccancer, colorectal cancer (CRC), epithelial carcinomas, brain cancer,endometrial cancer, testis cancer, cholangiocarcinoma, gallbladdercarcinoma, and hepatocellular carcinoma.

Biological samples are described herein, e.g., in the definition ofBiological Sample. In some embodiment, the biological sample is serum orof a tumor.

In embodiments involving detection of FGF19 and/or FGFR4 expression,FGF19 and/or FGFR4 polynucleotide expression and/or FGF19 and/or FGFR4polypeptide expression may be detected. In some embodiments involvingdetection of FGF19 and/or FGFR4 expression, FGF19 and/or FGFR4 mRNAexpression is detected. In other embodiments, FGF19 and/or FGFR4polypeptide expression is detected using an anti-FGF19 agent and/or ananti-FGFR4 agent. In some embodiments, FGF19 and/or FGFR4 polypeptideexpression is detected using an antibody. Any suitable antibody may beused for detection and/or diagnosis, including monoclonal and/orpolyclonal antibodies, a human antibody, a chimeric antibody, anaffinity-matured antibody, a humanized antibody, and/or an antibodyfragment. In some embodiments, an anti-FGF19 antibody described hereinis use for detection. In some embodiments, FGF19 and/or FGFR4polypeptide expression is detected using immunohistochemistry (IHC). Insome embodiments, FGF19 expression is scored at 2 or higher using anIHC.

In some embodiments involving detection of FGF19 and/or FGFR4expression, presence and/or absence and/or level of FGF19 and/or FGFR4expression may be detected. FGF19 and/or FGFR4 expression may beincreased. It is understood that absence of FGF19 and/or FGFR4expression includes insignificant, or de minims levels. In someembodiments, FGF19 expression in the test biological sample is higherthan that observed for a control biological sample (or control orreference level of expression). In some embodiments, FGF19 expression isat least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 75-fold, 100-fold, 150-fold higher, or higher in the testbiological sample than in the control biological sample. In someembodiments, FGF19 polypeptide expression is determined in animmunohistochemistry (“IHC”) assay to score at least 2 or higher forstaining intensity. In some embodiments, FGF19 polypeptide expression isdetermined in an IHC assay to score at least 1 or higher, or at least 3or higher for staining intensity. In some embodiments, FGF19 expressionin the test biological sample is lower than that observed for a controlbiological sample (or control expression level).

In some embodiments, FGF19 expression is detected in serum and FGFR4expression is detected in a tumor sample. In some embodiments, FGF19expression and FGFR4 expression are detected in a tumor sample. In someembodiments, FGF19 expression is detected in serum or a tumor sample,and FGFR4 downstream molecular signaling and/or FGFR4 expression isdetected in a tumor sample. In some embodiments, FGF19 expression isdetected in serum or a tumor sample, and Wnt pathway activation isdetected in a tumor sample. In some embodiments, FGF19 expression isdetected in serum or a tumor sample, and FGFR4 downstream molecularsignaling and/or FGFR4 expression and/or Wnt pathway activation isdetected in a tumor sample.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition(s)effective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody of the invention; and (b)a second container with a composition contained therein. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat a particular condition, e.g. cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES

The following materials and methods were used in Examples 1-12.

Gene Expression

Total RNA from frozen tissue samples was extracted using RNA STAT-60according to the manufacturer's protocol (Tel-test “B” Inc.). Total RNAfrom cultured cells was isolated with RNeasy kit using themanufacturer's protocol (Qiagen). The contaminating DNA was removedusing the DNA-free kit (Ambion; cat#1906)) and the samples were used forreal-time PCR. Specific primers and fluorogenic probes for human FGF19,FGFR4 and RPL19 mRNAs (Table 2) were designed using Primer Express 1.1(PE Applied Biosystems) and used to quantify gene expression. The genespecific signals were normalized to the signal of the RPL19 housekeepinggene. Triplicate sets of data were averaged for each condition.

TABLE 2 Forward Primer Reverser Primer Probe CYP7α1 CCATGATGCAAAACCTACCCAGACAGCGCTCT TGTCATGAGACCTCCGG CCAAT TTGA GCCTTCC (SEQ ID NO: 11)(SEQ ID NO: 12) (SEQ ID NO: 13) GAPDH AATTTGCCGTGAGTGG CAGTGGCAAAGTGGAGCCATCAACGACCCCTTC AGTC ATTGT ATTGACCTC (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) FGF19 AGACCCCAAGTCTTGT AATATCATGTTGGAAACCGCTGCTTCCACACAG CAATAAC ACCAAGTG CAA (SEQ ID NO: 17) (SEQ ID NO: 18)(SEQ ID NO: 19) FGFR4 GCTCTTGACGGGAGCA CGCCATTTGCTCCTGTGCAGGCTTCCAGCTTCT TT TT C (SEQ ID NO: 20) (SEQ ID NO: 21)(SEQ ID NO: 22) RPL19 AGCGGATTCTCATGGA CTGGTCAGCCAGGAGCTCCACAAGCTGAAGGCA ACA TT GACAAGG (SEQ ID NO: 23) (SEQ ID NO: 24)(SEQ ID NO: 25)

In Situ Hybridization

³³P-UTP labeled sense or antisense probes corresponding to human FGFR4(nucleotides 435 to 1183 of NM_(—)022963) or FGF19 (nucleotides 495 to1132 of NM 005117) were generated by polymerase chain reaction (Mauad etal. (1994) Am J Pathol 145, 1237-1245). Sections were deparaffinized,deproteinated in 4 mg/ml of proteinase K for 30 min at 37° C., andfurther processed for in situ hybridization (Holcomb et al. (2000) EmboJ 19, 4046-4055). Probes were hybridized to the sections at 55° C.overnight and unhybridized probes were removed by RNAse A treatments.The slides were dipped in NBT2 emulsion (Eastman Kodak), exposed for 4weeks at 4° C., developed and counterstained with hematoxylin and eosin.

Immunoprecipitation and Immunoblotting

Tissue samples (50 mg) were homogenized in 500 μl extraction buffer (20mM Tris pH 8, 137 mM NaCl, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1.5mM MgCl₂, complete protease inhibitor cocktail (Roche AppliedSciences)). Total proteins from cultured cells were extracted on ice for30 min with the extraction buffer. Lysates were centrifuged (10,000×g,15 min) and then cleared with Cibacron blue-agarose and ProteinG-agarose (GE Healthcare Life Sciences) overnight at 4° C. Lysates (100μg protein) were incubated in 1 ml PBS/0.1% Triton with 2 ug of theagarose coupled antibodies of interest for 1 h at 4° C. The gel slurrywas washed with the same buffer and eluted with 10 μl Elution buffer(Pierce Biotechnology). Samples were analyzed by Westerns blot using 2ug of biotinylated FGF19 antibody (BAF969; R&D systems), FGFR4 antibody(Genentech, Inc.) and IRDye 800 conjugated secondary reagents andvisualized using the Odyssey scanner (L1-Cor Biotechnology).

Immunohistochemistry

Formalin fixed paraffin embedded tissue sections were treated forantigen retrieval using Trilogy (Cell Marque) and then incubated with 10ug/ml FGF19 antibody (1D1; Genentech Inc). The immunostaining wasaccomplished using a biotinylated secondary antibody, an ABC-HRP reagent(Vector Labs) and a metal-enhanced DAB colorimetric peroxydase substrate(Pierce Laboratories).

Cell Migration Assay

The surface of 8 μm porosity 24-well format PET membrane filters (BDBiosciences) was coated overnight at 4° C. with 50 μl of type 1 collagen(50 μg/ml; Sigma) in 0.02 M acetic acid. Cells (5×10⁴) in serum freeminimal essential medium containing 0.1% BSA were added to the upperchamber. The lower chamber was filled with the same media and the plateswere incubated at 37° C. The next day the upper chamber was wiped with acotton swab and the cells that migrated to the lower side of the insertwere stained and counted under a microscope. Triplicate sets of datawere averaged for each condition.

Solid Phase Receptor Binding Assay

Maxisorb 96 well plates were coated overnight at 4° C. with 50 μl of 2μg/ml anti-human immunoglobulin Fcγ fragment specific antibody (JacksonImmunoresearch) and used to capture 1 μg/ml FGFR-Fc chimeric proteins (R& D Systems). The non-specific binding sites were saturated with PBS/3%BSA and FGF19 was incubated for 2 h in PBS/0.3% BSA in the presence ofglycosaminoglycans (Seikagaku Corporation) or oligosaccharides (NeoparinInc.). FGF19 binding was detected using a biotinylated FGF19 specificpolyclonal antibody (BAF969; R & D Systems) followed by streptavidin-HRPand TMB colorimetric substrate.

Receptor Pull Down Assay

FGFR-Fc chimeric proteins (400 ng) were incubated with 400 ng FGF19 or400 ng FGF1 and heparin (200 ng) in 50:50 Dulbecco's Modified EssentialMedia:Ham F12 containing 10 mM HEPES pH 7.4 and 0.1% BSA for 1 h.Protein G-agarose (20 μl) was added and further incubated for 30 min.The matrix was washed with PBS/0.1% Triton-X100, eluted with SDS-PAGEsample buffer containing reducing agent and analyzed by Western blotusing biotinylated FGF19 antibody (BAF969) or biotinylated FGF1 antibody(BAF232; R&D systems).

HSPG Solid Phase Binding Assay

Heparan sulfate proteoglycan (Sigma) was adsorbed to Maxisorb 96 wellplates overnight at 4° C. The non-specific binding sites were saturatedwith PBS/3% BSA and the wells were incubated with FGF19 or FGF1 (1:3serial dilutions from 1 ug/ml to 0.00017 ug/ml) (R & D Systems) for 1 h.The non-specific binding was determined in the presence of an excess ofheparin (10 ug/ml). The binding was detected with biotinylated specificantibodies and TMB colorimetric substrate. The specific binding wascalculated by subtracting the non-specific binding from the totalbinding.

Heparin Agarose Binding Assay

FGF19 and FGF1 protein (each at 400 ng/ml) were incubated with 20 μlheparin-agarose (GE Healthcare Life Sciences) in 50:50 Dulbecco'sModified Essential Media:Ham F12 containing 10 mM HEPES pH 7.4 and 0.1%BSA for 1 h. The gel slurry was washed with 1 ml of 20 mM Tris pH 7.4containing various NaCl concentrations and then with 1 ml of the samebuffer containing 20 mM NaCl. The bound proteins were eluted with SDSPAGE sample buffer containing reducing agent and analyzed by Westernblot.

Generation of FGF19 Monoclonal Antibodies

Balb/c mice were sequentially immunized with FGF19-His. In particular,Balb/c mice were immunized into each hind footpad 9 times (at two weekintervals) with 2.0 μg of hu FGF-19-His resuspended in MPL-TDM (RibiImmunochemical Research, Inc., Hamilton, Mont.). Three days after thefinal boost, spleens were harvested and popliteal lymph node cells werefused with murine myeloma cells P3X63Ag8.U.1 (ATCC CRL1597), using 35%polyethylene glycol. Hybridomas were selected in HAT medium. Ten daysafter the fusion, hybridoma culture supernatants were screened for mAbsbinding to the hu FGF-19 by ELISA. Cell lines producing antibodiesagainst human FGF-19 were cloned twice by limiting dilution. SelectedFGF19 antibody producing hybridomas were subcloned twice to insuremonoclonality. The clones were inoculated for ascites production andantibodies were purified by protein A-agarose affinity chromatography.

Total RNA was extracted from hybridoma cells producing the antibodies,using standard methods. The variable light (VL) and variable heavy (VH)domains were amplified using RT-PCR with the degenerate primers to heavyand light chain. The forward primers were specific for the N-terminalamino acid sequence of the VL and VH region. Respectively, the LC and HCreverse primers were designed to anneal to a region in the constantlight (CL) and constant heavy domain 1 (CH1), which is highly conservedacross species. Amplified VL and VH were cloned into mammalianexpression vectors. The polynucleotide sequence of the inserts wasdetermined using routine sequencing methods.

Analysis of Antibody Binding Affinity and Kinetics

For binding kinetics, Surface Plasmon Resonance (SRP) measurement with aBIAcore™-3000 was used (BIAcore, Inc., Piscataway, N.J.). Briefly,carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.) wereactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Anti-FGF19 or FGFR4 antibody was diluted with10 mM sodium acetate, pH 4.8, into 5 ug/ml before injected at a flowrate of 5 ul/minute to achieve approximately 500 response units (RU) ofcoupled antibody. Next, 1M ethanolamine was injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of eitherFGF19-His or FGFR4 molecules (0.7 nM to 500 nM) were injected in PBSwith 0.05% Tween 20 at 25′C at a flow rate of 25 ul/min. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated using asimple one-to-one Langmuir binding model (BIAcore Evaluation Softwareversion 3.2). The equilibrium dissociation constant (K_(d)) wascalculated as the ratio k_(off)/k_(on).

Antibody Epitope Excision

FGF19 protein (10 μg) was incubated for 2 h in 50 mM Tris, pH 7.4 with50 μl of agarose coupled-antibody. The resin was washed and digestedwith 0.1 μg trypsin (Promega) overnight at 37° C. in 100 mM ammoniumbicarbonate pH 8. The gel slurry was washed and the bound peptides wereeluted with 10% trifluoroacetic acid (TFA) and analyzed by MALDI-TOF-MS(Voyager; Applied Biosystems). Candidate peptides were subjected tocollision induced dissociation (QSTAR) and manually sequenced to confirmthe peptide mass mapping identification (FIG. S1).

Solid Phase Antibody Binding Assay

Non-specific binding sites of HisGrab Nickel coated plates (Pierce) weresaturated with PBS/3% BSA. The wells were incubated with 1 μg/mlFGF19-His in PBS/0.3% BSA for 1 h. The plates were washed and incubatedfor 1 h with FGF19 antibodies (at concentrations ranging from 1 ug/ml to0.000017 ug/ml) in the presence or the absence of FGF19 peptides inPBS/0.3% BSA. The bound antibodies were detected using a HRP conjugatedanti-mouse IgG (Jackson Immunoresearch) and the TMB peroxydasecolorigenic substrate (KPL).

CYP7α1 Expression Analysis

HEP3B cells were starved overnight in serum free Dulbecco's ModifiedEssential Media:Ham F12 (50:50) and treated with 100 ng/ml FGF19 for 6 hin the presence or the absence of antibodies 1A6, 1A1 or isotype-matchedcontrol antibody (each at concentrations ranging from 10 ug/ml to 0.04ug/ml). CYP7α1 expression was evaluated by semi-quantitative RT-PCRusing gene specific primers and probes (Taqman ABI PRISM 7700, AppliedBiosystems) and normalized to GAPDH expression. Triplicate sets of datawere averaged for each condition.

FGFR4/MAPK Phosphorylation

HEP3B cells starved overnight in serum free media were treated with 40ng/ml FGF19 for 10 mM in the presence or the absence of antibodies.Cells were lysed in R27A buffer (Upstate) supplemented with 10 mM NaF, 1mM sodium orthovanadate, and Complete protease inhibitor tablet (Roche).Lysates were prepared, electrophoresed and analyzed by Western blotusing phospho-FRS2, phospho-MAPK and MAPK specific antibodies (CellSignaling) and FRS2 specific antibody (Santa Cruz).

Xenograft Experiments

All animal protocols were approved by an Institutional Animal Care andUse Committee. Six- to eight-week-old athymic BALB/c female mice(Charles Rivers Inc.) were inoculated subcutaneously with 5×10⁶ cells(200 μl/mouse). After 7 days, mice bearing tumors of equivalent volumes(−100 mm³) were randomized into groups (n=10) and treatedintraperitoneally twice weekly. Tumors were measured with an electroniccaliper (Fowler Sylvac Ultra-Cal Mark III) and average tumor volume wascalculated using the formula: (W2×L)/2 (W, the smaller diameter; L, thelarger diameter). The statistical difference was analyzed using theStudent's t-test for normal distribution. Values of P<0.05 wereconsidered significant.

FGFR4, FRS2, and β-Catenin Phosphorylation in Xenograft Tumors

Tumors excised from control (gp120) and anti-FGF19 (1A6) antibodiestreated animals were homogenized in lysis buffer containing 50 mMTris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% sodiumdeoxycholate, 1 mM NaF, 1 mM sodium orthovanadate, and mini proteaseinhibitor tablet (Roche). Protein concentrations of the lysates weredetermined using the BCA protein assay reagent (Pierce). Equal amountsof proteins (100 μg protein) were incubated with 1 μg of anti-FGFR4antibody (clone 1G7; Genentech inc.) or anti-FRS2 (UpState) antibodyimmobilized onto protein A-Sepharose for 2 h at 4° C. with gentlerotation. Matrix was washed with lysis buffer and immunocomplexes elutedin 2× Laemmli buffer, boiled, and microcentrifuged. Proteins wereelectrophoresed on SDS-PAGE, transferred to nitrocellulose membrane, andprobed with phosphotyrosine antibody (1:1000 dilution, 4G 10, UpState).After washing and incubating with secondary antibody, immunoreactiveproteins were visualized by the ECL detection system (Amersham). ERK2phosphorylation levels were assessed without prior immunoprecipitationusing phospho-ERK2 antibody (1:1000 dilution, Santa Cruz Biotech) andβ-catenin phosphorylation was assessed without prior immunoprecipitationusing an antibody directed against N-terminally dephosphorylatedβ-catenin (1:1000 dilution, UpState). Membranes were stripped (Pierce)and reprobed with appropriate antibodies to determine total protein.

Micro-CT Imaging and Analysis of Hepatocellular Carcinomas in FGF19 TGMice

Liver tumors were identified by micro-ct imaging with Fenestra-LC, aliver specific contrast agent. Fenestra-LC is an iodinated triglyceridethat mimics chylomicron remnants and exploits endogenous lipid metabolicpathways resulting in hepatocyte contrast accumulation. These agentshave been previously been previously employed as means to identifyhepatic liver tumors. (Lee et al., 1997; Weichert et al., 1996) At6-months of age, FGF19 transgenic mice were injected with Fenestra LC(Advanced Research Technologies Inc. Saint-Laurent, Quebec, Canada), 20μl/g iv, and a conscious 3-hour hepatic uptake was allowed before micewere euthanized and livers resected for gross analysis, weighing,ex-vivo micro-CT analysis (μCT 40 system; Scanco Medical, Bassersdorf,Switzerland), and histological staining. Whole livers were lightlyblotted on gauze and submerged in soybean oil (Sigma-Aldrich, St. Louis,Mo.) in preparation for micro-CT imaging. For each liver, 90-minutescans were obtained at 30-μm isotropic voxel size, with 512 projectionsat an integration time of 300 ms, energy of 45 keV, and tube current of177 μA. Volumetric image files were analyzed using image analysissoftware from AnalyzeDirect (Lenexa, Kans.). An intensity threshold of−16 Houndsfield Units (HU) was used to segment the tissue mass from thebackground signal (soybean oil). A second threshold (26 HU) was employedto estimate hepatic tissue volumes associated with functional hepatictissue that accumulated the contrast agent resulting in hyper-intenseregions defining normal hepatic tissue. Hepatic tissues of FGF19transgenic mice where there was minimal attenuation due to smallcontrast agent concentrations, including vasculature, gall bladder andbiliary ducts, and hepatocellular carcinomatous lesions, appeared lessintense. An average of the total low-intensity hepatic volumes fromwild-type FVB mice, which did not have hepatocellular carcinomas, wassubtracted from both the FGF19 transgenic control and treated groups toobtain volumes associated only with tumors. Data are expressed as apercentage of tumor volume of total liver volume.

Statistical Analysis

Statistical significance was analyzed using the unpaired two-tailedStudent's t-test. Values of P<0.05 were considered significant. Data areexpressed as the mean±s.e.m.

The following materials and methods were used in Examples 13-17:

Cells

HCT116 cells (ATCC, Rockville, Md.) were routinely maintained at 37° C.and 5% CO₂ in RPMI 1640 containing 10% tetracycline-free fetal bovineserum and 4 mmol/L L-glutamine. Serum-starved cells were incubated witheither vehicle or FGF19 (25-100 ng/ml, 10 min). In separate experiments,cells were treated with either control antibody (gp120) or FGF19antibody (1A6, 10 μg/ml) for 3-24 hrs. To further evaluate the effectson β-catenin activation, cells were pretreated with a proteasomeinhibitor, MG132 (Biomol, Plymouth Meeting, Pa.) at 1 μM concentrationfor 4 hr followed by anti-FGF19 mab 1A6 treatment for 24 hrs to evaluatephosphorylation of Ser33/Ser 37, Ser 45 and Thr41 on β-catenin. Afterincubation, cells were washed in cold PBS and lysed for either proteinor RNA analysis.

Immunoprecipitation and Western Blot Analysis

Cells were lysed in modified RIPA buffer (50 mM Tris-Cl, pH 7.5; 150 mMNaCl; 1% IGEPAL; 1 mM EDTA; 0.25% sodium deoxycholate; 1 mM NaF; 1 mMNa₃VO₄; protease inhibitors cocktail (Sigma-Aldrich, St. Louis, Mo.) andclarified by centrifugation. Protein concentrations of the lysates weredetermined using the BCA protein assay reagent (Pierce, Rockford, Ill.).Equal amounts of proteins were incubated with specific antibodyimmobilized onto protein A-Sepharose (Sigma-Aldrich) for 2 hours at 4°C. with gentle rotation. Beads were washed extensively with lysis bufferand immunecomplexes were eluted in 2× Laemmli buffer, boiled andmicrocentrifuged. Proteins were resolved on SDS-PAGE, transferred tonitrocellulose membrane and incubated with specific primary antibodies.After washing and incubating with secondary antibodies, immunoreactiveproteins were visualized by the ECL detection system (Amersham,Arlington Ht. IL), The antibodies used for immunoprecipitation andimmunoblotting were anti-β-catenin mAbs from BD Transduction (San Diego,Calif.), anti-active-β-catenin antibody directed against N-terminallydephosphorylated β-catenin, anti-phosphotyrosine (4G10) andanti-E-cadherin antibody from UpState Biotech (Charlottesville, Va.),anti-phospho-β-catenin (Ser33/Ser37 and Ser45/Thr41 specific) antibodyfrom Cell Signaling (Danvers, Mass.), and anti-FGFR4 mAb (1G7)(Genentech, Inc.). Where indicated, the membranes were stripped (Pierce)and reprobed with another antibody. The densities of specific proteinbands were analyzed using Adobe Photoshop cs2 version 9 (Adobe Systems,Mountain View, Calif.). Quantitative analyses of tyrosine and Ser/Thrphosphorylation of β-catenin and E-cadherin were performed bydetermining the ratio between total protein and the phosphorylation byusing the data from three separate experiments.

Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS)

Indirect quantification of N-terminal β-catenin phosphorylation levelswas performed using linear ion trap mass spectrometry. β-catenin wasimmunoprecipitated from cells pretreated with MG132 followed bytreatment with control (gp120) or anti-FGF19 mab 1A6, and separatedusing Tris-Gly SDS PAGE. Gels were Coomassie stained and the β-cateninbands were cut out and reduced in 10 mM DTT for 30 min room temperatureand cysteines were alkylated with 50 mM iodoacetamide 15 min at roomtemperature before tryptic digestion. The peptides were digested intrypsin (10 ng/μl) in 50 mM sodium bicarbonate pH 8.0 and peptidemixtures (3 mL) were loaded onto a 0.25×30 mm trapping cartridge packedwith Vydac 214MS low-TFA C4 beads. This cartridge was placed in-linewith a 0.1×100 mm resolving column packed with Vydac 218MS C18 beads.The resolving column was constructed using a “picofrit” (New Objective)fused silica capillary pulled to a 15 mm metal-coated tip, which formeda micro-electrospray emitter. Peptides were eluted with 1 hour gradientsof acetonitrile containing 0.1% formic acid at a rate of 0.3 mL/min.Data dependent tandem mass spectrometry was performed using a linear iontrap instrument (LTQ; Finnigan). The Sequest database searching programwas used to generate cross correlation scores for each CID spectrum.Proteins matched by only a single peptide were confirmed by manualinterpretation of the collision-induced dissociation spectra.Phosphorylated peptides were manually confirmed. Peak areas were thenintegrated to determine relative abundance of peptides.

Wnt-Target Gene Expression Analyses

Total RNA was isolated using the Qiagen RNA isolation kit (Qiagen, CA)and DNase treated (Applied Biosystems, Foster City, Calif.) followingthe manufacturer's protocol. RNA concentration was determined usingND-1000 spectrophotometer (Wilmington, Del.). Real-time quantitative PCRwas performed to determine the relative abundance of Wnt-target gene(cyclin D1, CD44, E-cadherin, c-jun) mRNAs. Probes were labeled with FAM(5′ end) and TAMRA (3′ end). The primers and probe sequences were asfollows:

human cyclin D1 Forward: (SEQ ID NO: 26) GCT GCT CCT GGT GAA CAA GC;Reverse: (SEQ ID NO: 27) TGT TCA ATG AAA TCG TGC GG; Probe:(SEQ ID NO: 28) CAA GTG GAA CCT GGC CGC AAT GAC; human CD44 Forward:(SEQ ID NO: 29) GAA AAA TGG TCG CTA CAG CAT CT; Reverse: (SEQ ID NO: 30)GGT GCT ATT GAA AGC CTT GCA; Probe: (SEQ ID NO: 31)CGG ACG GAG GCC GCT GAC C; human E-cadherin Forward: (SEQ ID NO: 32)GAC TTG AGC CAG CTG CAC AG; Reverse: (SEQ ID NO: 33)GTT GGT GCA ACG TCG TTA CG; Probe: (SEQ ID NO: 34)CCT GGA CGC TCG GCC TGA AGT G; human c-jun Forward: (SEQ ID NO: 35)CGT TAA CAG TGG GTG CCA ACT; Reverse: (SEQ ID NO: 36)CCC GAC GGT CTC TCT TCA AA; Probe: (SEQ ID NO: 37)ATG CTA ACG CAG CAG TTG CAA ACA;Human specific ribosomal protein L-19 (RPL-19): Forward: (SEQ ID NO: 38)AGC GGA TTC TCA TGG AAC A; Reverse: (SEQ ID NO: 39)CTG GTC AGC CAG GAG CTT; Probe: (SEQ ID NO: 40)TCC ACA AGC TGA AGG CAG ACA AGG.

Amplification reactions (50 μl) contained 100 ng of RNA template, 5mmol/L of MgCl2, 1× buffer A, 1.2 mmol/L of dNTPs, 2.5 U of TaqGoldpolymerase, 20 U of RNase inhibitor, 12.5 U of MuLV reversetranscriptase, 2 mmol/L each forward and reverse primer, and 5 μmol/L ofprobe (Perkin Elmer). Thermal cycle (Perkin Elmer ABI Prism 7700sequence detector) conditions were 48° C. for 30 minutes, 95° C. for 10minutes, and 95° C. for 15 seconds, and 60° C. for 1 minute for 40cycles. Analyses of data were performed using Sequence Detector 1.6.3(PE Applied Biosystems) and results for genes of interest werenormalized to the RPL19 gene.

ShRNA Studies

The pHUSH inducible vector system comprising a shRNA expression shuttleplasmid and a viral vector backbone containing a TetR-IRES-Puro cassettewas used (Hoeflich, K P et al, Cancer res 66:999-1006 (2006)). FGFR4knockdown vectors were constructed by designing custom siRNA sequences,converting them into shRNA, and testing their efficacy in transientco-transfection experiment in 293T cells. The following shRNA sequenceswere cloned into pShuttle-H1 and then H1-shRNA cassette was transferredinto pHUSH-GW by a Gateway (Invitrogen, Carlsbad, Calif.) recombinationreaction:

FGFR4 shRNA2 Forward: (SEQ ID NO: 41)GAT CCC CCC TCG TGA GTC TAG ATC TAT TCA AGA GATAGA TCT AGA CTC ACG AGG TTT TTT GGA AA; Reverse: (SEQ ID NO: 42)AGC TTT TCC AAA AAA CCT CGT GAG TCT AGA TCT ATCTCT TGA ATA GAT CTA GAC TCA CGA GGG GG; FGFR4 shRNA5 Forward:(SEQ ID NO: 43) GAT CCC CGA ACC GCA TTG GAG GCA TTA TCA AGA GAAATG CCT CCA ATG CGG TTC TTT TTT GGA AA; Reverse: (SEQ ID NO: 44)AGC TTT TCC AAA AAA GAA CCG CAT TGG AGG CAT TTCTCT TGA TAA TGC CTC CAA TGC GGT TCG GG; hEGFP control Forward:(SEQ ID NO: 45) GAT CCC CGC AGC ACG ACT TCT TCA AGT TCA AGA GACTTG AAG AAG TCG TGC TGC TTT TTT GGA AA; Reverse: (SEQ ID NO: 46)AGC TTT TCC AAA AAA GCA GCA CGA CTT CTT CAA GTCTCT TGA ACT TGA AGA AGT CGT GCT GCG GG.All constructs were verified by sequencing.

Generation of Inducible-shRNA Cell Clones

HCT116 cells were transfected using LipofectAmine 2000 plus(Invitrogen). As the puromycin resistance gene encoded in the vector isunder the control of a constitutive β-actin promoter, 5 μg/mL puromycinwas used to select transfected cells expressing shRNA. Stable cloneswere isolated, treated with 1 μg/mL doxycycline (BD Clontech, San Jose,Calif.) for 7 days to induce expression of siRNA. Functional FGFR4protein knockdown was assessed by Western blotting.

Statistical Analysis

Student's two-tailed t test was used to compare data between two groups.One-way analysis of variance and Dunnett's test were used to comparedata between three or more groups. P-value <0.05 was consideredstatistically significant.

Example 1 Analysis of FGF19 and FGFR4 Expression in Human Tissues

FGF19 and FGFR4 protein expression was evaluated in human colonadenocarcinomas, lung squamous cell carcinomas (SCC), and hepatocellularcarcinomas (HCC). FGF19 was overexpressed in 6 out of 10 colonadenocarcinomas (FIG. 2A) and in 7 out of 10 lung SCC relative to normaltissues (FIG. 2B). Compared to normal tissues, FGFR4 expression was notsignificantly altered in colon tumors but appeared downregulated in SCC(FIGS. 2A and 2B).

To localize FGF19 and FGFR4 mRNA expression in tumor tissues, weperformed in situ hybridization. Messenger RNA for both genes wasprominent in neoplastic epithelial cells in colon adenocarcinomas andlung SCC (FIGS. 2C and D). In a tissue microarray comprised of 35 colonadenocarcinoma cases, 26 (74%) had positive signal for FGF19 mRNA and 27(77%) had positive signal for FGFR4 mRNA. Treatment with anti-FGF19antibody targets both non-tumor-derived FGF19 and tumor-derived FGF19,and thus anti-FGF19 treatment may have clinical benefit inFGFR4-positive tumors that lack FGF19 expression. Table 3 shows thepresence or absence of co-expression of FGFR4 and/or FGF19 mRNAs in thecolon adenocarcinoma tissue microarray samples:

TABLE 3 Colon Adenocarcinoma FGFR4+ FGFR4− FGF19+ 21 (60%) 5 (14%)FGF19−  6 (17%) 3 (9%) Overlap between the presence of FGF19 and FGFR4 in colon adenocarcinomaswas observed in a majority of tumor samples. Of 14 lung SCC cases, 14(100%) had positive signal for FGF19 mRNA and 13 (93%) had positivesignal for FGFR4 mRNA. In addition, neoplastic epithelial cells showedstrong FGF19 protein staining by immunohistochemistry in both colonadenocarcinomas (FIG. 2C) and lung SCC (FIG. 2D). These relatively highexpression frequencies suggested a significant overlap between thepresence of FGF19 and FGFR4 in lung SCCs. Because systemic FGF19expression in transgenic mice promotes hepatocellular carcinomas (HCC)(Nicholes et al., 2002), we also evaluated FGF19 and FGFR4 mRNAexpression in liver samples. Of 50 cases of hepatocellular carcinoma, 23(46%) demonstrated positive signal for FGF19 mRNA and 30 (60%) for FGFR4mRNA. Both genes were expressed in the neoplastic hepatocytes(representative examples shown in FIG. 2E). The neoplastic hepatocytesalso showed strong FGF19 protein staining by immunohistochemistry (FIG.2E). Table 4 shows the presence or absence of co-expression of FGFR4and/or FGF19 mRNAs in the colon adenocarcinoma tissue microarraysamples:

TABLE 4 HCC FGFR4+ FGFR4− FGF19+ 21 (41%) 4 (8%) FGF19− 11 (22%) 15(29%)Overlap between FGFR4 and FGF19 expression was observed in a largepercentage of samples.

These results showed that FGF19 and FGFR4 are expressed in several typesof human cancers.

Because cirrhosis often precedes hepatocellular carcinoma, FGF19 mRNAexpression was evaluated in cirrhotic liver. These samples showed strongFGF19 mRNA and protein signals in regenerative nodule hepatocytes (FIG.2F), suggesting that FGF19 expression occurs early during liverneoplastic progression.

FGF19 mRNA expression was also evaluated in several types of primaryepithelial tumors by in situ hybridization. FGF19 mRNA expression wasdetected in 16/38 (42%) cases of breast adenocarcinoma, 39/70 (56%)cases of ovarian adenocarcinoma, and 8/79 (10%) cases of pancreaticadenocarcinoma. These results showed that FGF19 mRNA was expressed inseveral types of primary tumor. In addition, a panel of colonadenocarcinoma was screened for expression of FRFR4 protein usingimmunohistochemistry, and 18/20 samples were positive for FGFR4expression.

Example 2 FGF19 and FGFR4 are Expressed in Human Tumor Cell Lines andXenograft Tissues

FGF19 and FGFR4 mRNA and protein expression was analyzed in a panel ofcolon, breast, and liver tumor cell lines. We found FGF19 mRNAexpression in a subset of colon cancer cell lines, including Colo201,Colo205, SW620, SW480, and HCT116 (FIG. 3A). SNU185, SNU398, MCF7 andall colon cancer cell lines tested expressed FGFR4 mRNA. FGF19 and FGFR$protein expression was determined in a panel of cancer cell lines usingwestern blot analysis. With the exception of HT29, which did not expressFGF19 protein, the FGF19 and FGFR4 protein levels agreed with their mRNAexpression in these cell lines (FIG. 3B). The electrophoretic mobilityof the FGF19 secreted by the cell lines was consistent with the expectedmolecular mass of 24 kDa. However, additional lower molecular mass bandswere also detected, possible representing truncated protein.

To verify that FGF19 protein expression is maintained in vivo, coloncancer cell line-derived tumor xenografts were evaluated byimmunohistochemistry (FIGS. 3C and 3D). Colo205 xenograft tumor tissuehad strong FGF19 expression in all neoplastic epithelial cellsthroughout the tumor, but not in the associated mouse stroma or adjacentnormal tissue. SW620 and HCT116 xenografts showed positiveimmunoreactivity in scattered neoplastic cells. Immunohistochemistry ofthe FGF19 negative HT29 cell line xenograft did not show any staining.These results suggested that colon cancer cell lines express FGF19 upongrowing in vitro in culture dishes as well as in vivo in a subcutaneousxenograft setting.

Example 3 FGF19 is not a Heparin-binding Factor

Glycosaminoglycan binding assays were performed to directly assesswhether FGF19 protein and heparin interact. In a solid phase bindingassay, FGF1 demonstrated a dose dependent binding to thesurface-adsorbed purified heparan sulfate proteoglycan (FIG. 4A). Bycontrast, FGF19 did not bind the coated material. In a pull-down assay,FGF1 strongly bound to heparin-agarose affinity matrix. As previouslyreported FGF1 was desorbed only with buffers containing NaClconcentrations higher than 1M. By contrast, FGF19 did not significantlybind to heparin agarose at the lowest concentration of NaCl tested (20mM), and no protein could be detected after washes with higher NaClconcentrations (FIG. 4B). Together, these results indicate that FGF19did not bind significantly to glycosaminoglycan and therefore can not beconsidered a heparin binding factor.

Example 4 FGF19 Specifically Binds to FGFR4

Previous co-immunoprecipitation studies suggest that the receptorbinding specificity of FGF19 is restricted to FGFR4 (Xie et al., 1999).To examine FGF19's binding specificity more completely we assessed itsinteraction with all known human FGFRs in their different alternativelyspliced forms including the recently identified FGFR5 (FGFR1L) (Sleemanet al., 2001). In a solid phase assay FGF19 dose dependent binding wasrestricted to FGFR4 (FIG. 4C). In a receptor pull down assay FGF1 boundto all FGFRs whereas FGF19 interaction was limited to FGFR4 (FIG. 4D).These results are consistent with previous findings (Ornitz et al.,1996; Xie et al., 1999) and further emphasize the unique bindingspecificity of FGF19 for FGFR4.

Example 5 FGF19 Binding to FGFR4 is Modulated by Glycosaminoglycan

The specificity of the glycosaminoglycan requirement for FGF19 bindingto FGFR4 was analyzed using a solid phase receptor binding assay. Asseen in FIG. 4E, heparin constituted the most potent promoter of FGF19interaction with FGFR4 (EC₅₀=0.0025 μg/ml), followed by heparan sulfate(EC₅₀=0.9 μg/ml), chondroitin sulfate B (EC₅₀=1 μg/ml) and chondroitinsulfate A (EC₅₀=4 μg/ml). Chondroitin sulfate C did not promote FGF19binding to FGFR4.

The effect of various lengths of heparin polysaccharide on FGFR4 bindingpromotion was also analyzed. The heparin octasaccharide showed only aminimal effect on FGFR4 binding at the highest concentration (10 μg/ml)(FIG. 4F). The dose dependent promotion of FGF19 binding to FGFR4 wasseen with heparin decasaccharide and longer fragments and this effectwas proportional to the heparin molecular weight. Taken together, theseresults showed that heparin constituted the most potentglycosaminoglycan supporting FGF19 binding to FGFR4, and that heparin'sactivity was proportional to its molecular weight.

Example 6 FGF19 Binds to FGFR4 with High Affinity

FGF19 binding affinity to FGFR4 was assessed by incubating increasingconcentration of [¹²⁵I]FGF19 with immobilized FGFR4 and heparin in thepresence or the absence of an excess of unlabeled ligand. FGF19demonstrated a dose-dependent and saturable binding to FGFR4 (FIG. 4G,inset). A K_(D) of 0.25 nM for the FGF19 binding to FGFR4 was determinedusing Scatchard analysis, confirming that the ligand and receptorinteract with high affinity.

Example 7 Generation of Anti-FGF19 Monoclonal Antibody

A panel of anti-FGF19 mouse monoclonal antibodies was generated asdescribed above. The polynucleotide sequence of the inserts wasdetermined using routine sequencing methods. The anti-FGF19 mab 1A6 VLand VH amino acid sequences are shown in FIG. 1.

Example 8 Analysis of Anti-FGF19 Monoclonal Antibody Binding AffinityUsing Surface Plasmon Resonance and Enzyme-linked Immunosorbent Assays

To determine the binding affinity of mouse anti-FGF19 Mabs, surfaceplasmon resonance (SRP) measurement was performed with a BIAcore™-3000was used (BIAcore, Inc., Piscataway, N.J.) as described above. Theresults of this analysis are shown in Table 5.

TABLE 5 Antibody Kd Kon Koff 1A6 (anti-FGF19)   <9 pM 5.6 × 10⁵ (M⁻¹s⁻¹) <5 × 10⁻⁶ (s⁻¹) 1D1 (anti-FGF19)   32 nM 2.4 × 10⁴ (M⁻¹s⁻¹) 7.7 × 10⁻⁴(s⁻¹) 1A1 (anti-FGF19) ~300 nM   1 × 10⁶ (M⁻¹s⁻¹)   3 × 10⁻² (s⁻¹)

In enzyme-linked immunosorbent assays, anti-FGF19 mabs 1A1 and 1A6 boundto FGF19 with a comparable EC50 of 40 pM whereas anti-FGF19 mab 1D1bound with an EC50 of 400 pM (FIG. 5A). In a solid phase receptorbinding assay, 1A6 blocked FGF19 binding to FGFR4 with an IC50 of 3 nM(FIG. 5B). 1A1, 1D1 and an irrelevant control antibody did not inhibitthis interaction.

Example 9 Anti-FGF19 Antibody Blocked FGF19 Signaling in a Cell-basedAssay

Several cell-based assays were performed in order to determine whetherthe anti-FGF19 antibodies blocked the interaction of FGF19 and FGFR4.

FGF19 plays a role in cholesterol homeostasis by repressing hepaticexpression of cholesterol-7-α-hydroxylase 1 (Cyp7α1), the rate-limitingenzyme for cholesterol and bile acid synthesis (Gutierrez et al (2006)Arterioscler Thromb Vasc Biol 26, 301-306; Yu et al (2000) J Biol Chem275, 15482-15489). The ability of anti-FGF19 antibodies 1A1 and 1A6, orisotype-matched negative control antibody (at concentrations rangingfrom 10 ug/ml to 0.04 ug/ml) to block FGF19-induced downregulation ofcyp7α1 was assessed using hepatocellular carcinoma HEP3B cells(Schlessinger, Science 306:1506-1507 (2004)) as described above. In theabsence of anti-FGF19 antibody, FGF19 treatment reduced cyp7α1expression by 75% (FIG. 5D). Treatment with 1.1 μg/ml mouse anti-FGF19Mab 1A6 abolished FGF19-induced repression of cyp7α1 expression. Bycontrast, treatment with mouse anti-FGF19 Mab clone 1A1 only reduced therepression by 50% at the highest concentration tested (10 μg/ml), butnot at lower antibody concentrations. The presence of a control antibodydid not affect FGF19 activity. The IC50 for anti-FGF19 antibody 1A6inhibition of FGF19-induced downregulation of cyp7α1 gene expression wasabout 0.4 ug/ml. The IC50 for anti-FGF19 antibody 1A1 inhibition ofFGF19-induced downregulation of cyp7α1 gene expression was about 10ug/ml.

Anti-FGF19 mab 1A6 was also tested for its ability to block theFGF19-induced FGF pathway activation in hepatocellular carcinoma Hep3Bcells (Eswarakumar et al (2005) Cytokine Growth Factor Rev 16:139-149;Schlessinger, J (2004) Science 306:1506-1507). Serum starvedhepatocellular carcinoma Hep3B cells were treated with FGF19 in theabsence or the presence of a negative control antibody or with variousconcentrations of anti-FGF19 monoclonal antibodies 1A6 or 1D1 (at 30, 10and 3.3 ng/ml), and FRS2 and MAPK phosphorylation determined asdescribed above. Treatment with anti-FGF19 Mab 1A6 significantly blockedFGF19-induced FRS2 and MAPK phosphorylation at all doses tested (FIG.5C). By contrast, treatment with the control antibody and anti-FGF19 mab1D1 did not show significant inhibitory activity.

Because FGFR4 plays a role in cell migration, we evaluated thechemotactic activity of FGF19 (Wang et al (2005) Clin Cancer Res10:6169-6178). In a modified Boyden chamber assay, FGF19 promoted HCT116cell migration in a dose dependent fashion, reaching a maximum at 16ng/ml (FIG. 10). The anti-FGF19 mabs were tested for the ability toinhibit FGF19-promoted cell migration. At 0.1 μg/ml, treatment withanti-FGF19 mab 1A6 inhibited FGF19-induced cell migration (FIG. 5E).Treatment with higher concentrations of 1A6 reduced cell migration tobelow the basal HCT116 cell migration level, likely by inhibiting bothexogenously added and endogenously produced FGF19.

These results demonstrated that anti-FGF19 antibody 1A6 was a potentinhibitor of FGF19 activity in vitro.

Example 10 Antibody 1A6 Binding Determinant is Localized in the FGF19Binding Interface with FGFR4

A mass spectrometric approach was used to localize the epitopes ofanti-FGF19 Mab 1A6 and 1A1 and to evaluate whether FGF19 conformationalcomponents contributed to their binding. We first attempted to isolatean epitope containing peptide from an FGF19 tryptic digest using anagarose-coupled 1A6 affinity matrix. This approach was unsuccessfulpossibly because the FGF19 fragmentation compromised the conformationalintegrity of 1A6 epitope. To test this hypothesis, we modified theprocedure and FGF19 was incubated with the agarose-coupled antibodiesand the adsorbed protein was then digested with trypsin. The analysis ofthe total digest demonstrated a complete fragmentation of the adsorbedFGF19, without 1A6 masking of any trypsin cleavage sites (FIG. 6A). Thematrix was washed extensively and the bound peptides were eluted andidentified by mass spectrometry. The non-specifically adsorbed peptideswere identified using an irrelevant control antibody coupled to agarosein a parallel experiment.

The results of this analysis are shown in FIG. 6. The agarose coupledanti-FGF19 mab 1A6 specifically recognized the FGF19 peptide G133-R155(FIGS. 6B and 8A). Agarose-coupled anti-FGF19 mab 1A1 specificallyrecognized the peptide G156-R180 (FIGS. 6C and 8B).

Because conjugation of anti-FGF19 mab 1D1 to agarose abolished itsbinding to FGF19, a peptide competition binding assay was used toidentify its epitope. Only FGF19 amino acids A183-G192 competed with mab1D1 binding (FIG. 6D). This peptide did not compete the binding of mab1A1 to FGF19. Because overlapping peptides were used in this competitionassay, we surmise that the mab 1D1 epitope is located in the last 4distal FGF19 amino acids (SFEK).

The epitopes of mabs 1A1, 1D1 and 1A6 were mapped onto the previouslydescribed structural model of FGF19 interaction with FGFR4 (Harmer et al(2004) Biochemistry 43, 629-640). The epitopes of 1A1 and 1D1 arelocated in a distal portion of FGF19 that is not represented on thismodel due to its lack of ordered structure. However, the 1A6 epitope islocalized in the FGF19 binding interface with FGFR4 (FIG. 6E). Theseresults suggest that anti-FGF19 Mab 1A6 directly occludes thereceptor-binding site of FGF19.

Example 11 Treatment with Anti-FGF19 Monoclonal Antibodies InhibitedTumor Growth in vivo

To determine whether FGF19 neutralization could inhibit tumor growth invivo, anti-FGF19 antibodies were tested in two tumor xenograph models asdescribed above. The colon cancer cell lines HCT116 and colo201 wereselected because they expressed both FGF19 and FGFR4 (FIG. 3A) and formtumors in vivo. In addition, anti-FGF19 antibody 1A6 showed a blockingactivity on the FGF19-induced HCT116 cells migration in vitro.

Mice with established HCT116 xenograft tumors were treated twice weeklywith 5 mg/kg of either anti-FGF19 mab 1A6 or a control antibody. At day35, treatment with anti-FGF19 mab 1A6 significantly suppressed tumorgrowth by 57% (p=0.07, n=5) compared to the control antibody treatedgroup (FIG. 7A). This study was repeated using a higher dose of antibody1A6 (15 mg/kg; 2× week) and a statistically significant suppression oftumor growth was observed (60% growth inhibition, p=0.01, n=5).

To verify that anti-FGF19 mab 1A6 inhibited tumor growth by blockingFGF19 activity, tumors were examined at the end of the study for markersof FGFR4 signaling. Activation of FGFR4, FRS2, ERK and β-catenin wassignificantly decreased in tumors from animals treated with anti-FGF19mab 1A6 compared to animals treated with the control antibody (FIG. 7B).

Next, we used xenografts of Colo201, a colon cancer cell line expressinghigher FGF19 levels than HCT116 (FIG. 1A). Treatment (30 mg/kg; 2×/week)with anti-FGF19 mab 1A6 significantly suppressed the growth ofestablished colo201 tumors (at day 27, 64% growth inhibition, p=0.03,n=5) compared to the control antibody (FIG. 7C). Analysis of the excisedtumors showed that treatment with anti-FGF19 mab 1A6 significantlydecreased FGFR4, FRS2, ERK and β-catenin activation in xenograft tumorscompared to the control antibody treatment (FIG. 7D). These resultsdemonstrated efficacy of anti-FGF19 mab 1A6 in colon cancer models anddemonstrated its activity with inhibition of FGF19 dependent FGFR4,FRS2, ERK and β-catenin activation.

Example 12 Treatment with Anti-FGF19 Monoclonal Antibodies PreventedHepatocellular Carcinomas and Weight Loss in FGF19 Transgenic Mice

Over-expression of FGF19 in the skeletal muscle of transgenic miceresulted in development of hepatocellular carcinomas by 10-12 months ofage (Nicholes et al., Am J Pathol. 160:2295-2307, 2002). To confirm thatFGF19 is acting as a tumor promoter in this model, we treated the FGF19transgenic mice with a tumor initiator, diethylnitrosamine (DEN), whichaccelerated tumor formation by 50%. To determine whether anti-FGF19 mab1A6 could prevent hepatocellular carcinomas, DEN-accelerated FGF19transgenic mice were treated with either 1A6 or control antibody(anti-gp120) for 6 months. At the end of the treatment, all of thecontrol-treated mice had grossly evident multifocal, largehepatocellular carcinomas throughout the liver lobes whereas anti-FGF19mab 1A6-treated animals had either no liver tumors or, in one case(#1862), a single small tumor present on the diaphragmatic surface ofthe median lobe (FIG. 9A). Liver weights from anti-FGF19 Mab 1A6-treatedmice (mean=1.71±0.05 grams) were significantly lower than liver weightsfrom control treated mice (mean=3.15±0.58 grams; p=0.014), but were notsignificantly different from those of normal FVB wild-type mice(mean=1.56±0.08 grams; p=0.82). In addition, tumor volume was determinedby micro-CT image analysis, corrected for tumor volume with normal FVBliver, and graphed as a percent of total liver volume (FIG. 9B). Percenttumor volume of anti-FGF19 Mab 1A6-treated mice (mean=7.5±3.2%) wassignificantly lower than control gp120-treated mice (mean=23.8±6.8%;p=0.05). Furthermore, tumor weights strongly correlated with percenttumor volume (r²=0.993702). These data clearly demonstrated thatanti-FGF19 Mab 1A6 effectively neutralized circulating FGF19 to preventtumor formation in FGF19 transgenic mice.

Because FGF19 causes weight loss when overexpressed as a transgene inmice (Tomlinson et al. Endocrinology. 2002 May; 143(5): 1741-7), weevaluated body weights of mice in the two treatment groups. Mice wereweighed weekly and body weights were compared between treatment groups.At 3 months of age, control treated mice (mean weight 27.98 g±0.8351;N=5) weighed significantly less than anti-FGF19 mab 1A6 treated (meanweight21.32±0.5036; N=6) (p<0.0001). Weights of 1A6-treated FGF19 TGmice were similar to weights of normal FVB wild-type mice that wereevaluated in a different experiment (mean weight 33.22±1.838 N=6). Thesedata demonstrated that treatment with anti-FGF19 mab 1A6 effectivelyabrogated FGF19-induced weight loss in FGF19 transgenic mice.

Example 13 FGF19 Treatment Induced Tyrosine Phosphorylation of β-Cateninand Caused Loss of E-cadherin Binding to Beta-catenin in HCT116 Cells

Hepatocellular carcinomas found in FGF19-expressing transgenic mice haveneoplastic cell that show immunoreactivity with beta-cadherin (β-cateninor b-cat) antibodies (Nicoles et al., supra). Furthermore, it has beensuggested that Wnt signaling can initiate or promote FGF signaling invarious cell types and organs during a variety of cellular processes,including human colorectal carcinogenesis, and that co-activation of Wntand FGF signaling pathways in tumors leads to more malignant phenotypes(see refs 7-12 in Cancer Biol & Therapy 5:9, 1059-64, 2006). Thus, theeffect of FGF19 or inhibition of FGF19 signaling on the Wnt signalingpathway was tested using treatment with FGF19, treatment with anti-FGF19monoclonal antibody 1A6, or FGFR4-directed shRNA knockdown in humancolon cancer (HCT116) cells. β-catenin tyrosine phosphorylation,β-catenin-E-cadherin binding and active-β-catenin levels were assessedin treated cells using immunoprecipitation and immunoblot analysis.

Treatment of colon cancer cells (HCT116) with FGF19 (25-100 ng/ml)resulted in a significant increase in tyrosine phosphorylation ofβ-catenin as early as 10 min (FIG. 11) when compared with vehicletreated controls. β-catenin binding to cadherins to form stablecell-cell adhesions has been shown to be regulated by tyrosinephosphorylation of β-catenin. Therefore, we evaluated E-cadherin levelsin cells treated with FGF19 by stripping and reprobing the tyrosinephosphorylation blot using anti-E-cadherin antibody. The results showeda substantial loss of E-cadherin binding to β-catenin in FGF19-treatedcells. Similar results were obtained when E-cadherin wasimmunoprecipitated and immunoblot analysis was performed usinganti-β-catenin antibody. The reduction in E-cadherin binding wasinversely proportional to the increased tyrosine phosphorylation levelsobserved in FGF19-treated cells.

Example 14 Inhibition of FGF19 Using Anti-FGF19 Antibody 1A6 ReducedActive-β-catenin Levels in HCT116 Cells

Previous studies have established that Wnt regulated β-catenindegradation is essential for carcinogenesis (Polakis et al., Genes Dev14:1837-51, 2000) and that Wnt signals are transmitted throughN-terminally dephosphorylated β-catenin (Staal F J T et al, EMBO Reports3:63-68, 2002). Using a specific antibody specific for β-catenindephosphorylated at residues Ser37 and Thr41, we examined whether FGF19or inhibition of FGF19 affects Wnt-signaling in HCT116 cells. Treatmentof HCT116 cells with FGF19 did not affect active-β-catenin levels at anydose or time point, indicating that endogenous FGF19 activated β-cateninat saturated levels in an autocrine fashion. However, treatment ofHCT116 cells with anti-FGF19 antibody 1A6 significantly reducedactive-β-catenin levels at timepoints as early as 3 hrs followingtreatment, and sustained decreased active-β-catenin levels for up to 24hrs (71.8±1.5% decrease vs gp120, p<0.001.) when compared with controlantibody (gp120) treated cells (FIG. 12).

Example 15 Treatment with Anti-FGF19 Antibody Induced Ser33/Ser37/Ser45and Thr41 Phosphorylation

Since FGF19 inhibition reduced active-β-catenin levels in HCT116 cells,we next evaluated whether treatment with anti-FGF19 antibody resulted inincreased N-terminal Ser-Thr phosphorylation of β-catenin and thustargeted β-catenin for ubiquitination and proteasomic degradation.HCT116 cells pretreated with a proteasome inhibitor (MG132, 1 μM) for 4hrs followed by treatment with anti-FGF19 monoclonal antibody 1A6 showeda significant increase in Ser33/Ser37 and Ser45/Thr41 phosphorylationwhen compared with proteasome inhibitor plus control antibody (gp120)treated cells (FIG. 13). Quantification of Ser33/37 phosphorylation (asdetermined by calculating the ratio between the total β-catenin proteinand phosphorylated protein level) showed a 123.4±7% increase (p<0.05) inanti-FGF19 antibody 1A6-treated cells vs control anti-gp120antibody-treated cells. Similarly Ser45/T41 phosphorylation wasincreased by 166.8±11% in anti-FGF19 monoclonal antibody 1A6-treatedcells vs control anti-gp120 antibody treated cells (p<0.05).

Ser-Thr phosphorylation in the N-terminus of β-catenin was furtheranalyzed using linear ion trap mass spectrometry. Signal intensities ofnon-phosphorylated β-catenin peptide were determined using linear iontrap mass spectrometry in cells pretreated with a proteasome inhibitorfollowed by treatment with either anti-FGF19 antibody 1A6 or controlanti-gp20 antibody. The data was normalized to non-related peptides(containing all 4 phosphorylation sites) that showed no difference insignal intensities from the treated and untreated samples. The β-cateninpeptide isolated from anti-FGF19 antibody 1A6-treated cells showed lowersignal intensity when compared with β-catenin peptide isolated fromcontrol anti-gp120 antibody-treated cells (FIG. 14), clearly indicatingincreased phosphorylation on the N-terminus of β-catenin in anti-FGF19monoclonal antibody 1A6-treated cells.

Example 16 Reduction of FGFR4 Expression Using shRNA Resulted in ReducedActive-β-catenin Levels

To determine whether inhibition of the FGFR4 receptor would mimic theeffect of FGF19 inhibition resulting from treatment with anti-FGF19antibodies, stable cell lines expressing FGFR4-directed shRNA andcontrol EGFP-directed shRNA were generated as described above. Thestable cell line expressing FGFR4-directed shRNA showed effectiveknock-down of FGFR4 protein expression. Immunoblot analysis of celllysates from a stable cell line expression FGFR4-directed shRNA showedalmost complete reduction of active-β-catenin levels when compared witha control stable cell line expressing shRNA directed to EGFP (FIG. 15).

Example 17 Treatment with Anti-FGF19 Antibody Reduced Wnt-target GeneTranscription Levels in Colon Cancer Cells

Wnt-target gene (cyclin D1, CD44, E-cad, c-jun) expression levels weredetermined using real-time PCR in anti-FGF19 antibody 1A6-treated HCT116cells. As shown in FIG. 16, treatment with anti-FGF19 antibody 1A6reduced cyclin D1, CD44, E-cad and c-jun mRNA expression levels at 6 hrswhen compared with expression of those genes in control antibody(anti-gp120)-treated cells.

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Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

1. A method of inhibiting relapse tumor growth, the method comprisingadministering an effective amount of an anti-FGF19 antibody to anindividual in need of such treatment, wherein the antibody comprises:(a) a light chain comprising (i) HVR (hypervariable region)-L1comprising SEQ ID NO:1; (ii) HVR-L2 comprising amino acids 50-56 of SEQID NO:4; and (iii) HVR-L3 comprising SEQ ID NO:3; and (b) a heavy chaincomprising (i) HVR-H1 comprising SEQ ID NO:5; (ii) HVR-H2 comprising-SEQID NO:6; and (iii) HVR-H3 comprising SEQ ID NO:7; wherein the relapsetumor growth is a hepatocellular carcinoma tumor growth or colorectalcancer tumor growth.
 2. A method of inhibiting relapse tumor growth, themethod comprising administering an effective amount of an anti-FGF19antibody to an individual in need of such treatment, wherein theantibody competes for binding to human FGF19 with an antibody of claim1, wherein the relapse tumor growth is a hepatocellular carcinoma tumorgrowth or colorectal cancer tumor growth.
 3. The method of inhibitingrelapse tumor growth of claim 2, wherein the relapse tumor growth is ahepatocellular carcinoma tumor growth.
 4. The method of inhibitingrelapse tumor growth of claim 2, wherein the relapse tumor growth is acolorectal cancer relapse tumor growth.
 5. The method of inhibitingrelapse tumor growth of claim 2, further comprising administering to thesubject an effective amount of a second medicament, wherein theanti-FGF19 antibody is a first medicament.
 6. The method of inhibitingrelapse tumor growth of claim 1, wherein a full length IgG form of theantibody specifically binds human FGF19 with a k_(on) of 6×10⁵ (M⁻¹s⁻¹)or better.
 7. The method of inhibiting relapse tumor growth of claim 1,wherein a full length IgG form of the antibody specifically binds humanFGF19 with a k_(off) of 5×10⁻⁶ (s⁻¹) or better.
 8. The method ofinhibiting relapse tumor growth of claim 1, wherein a full length IgGform of the antibody specifically binds human FGF19 with a bindingaffinity of 40 pM or better.
 9. The method of treating inhibitingrelapse tumor growth of claim 1, wherein the full length IgG form of theantibody specifically binds human FGF19 with a binding affinity of 20 pMor better.
 10. The method of inhibiting relapse tumor growth of claim 1,wherein the antibody inhibits FGF19 promoted cell migration.
 11. Themethod of inhibiting relapse tumor growth of claim 1, wherein theantibody inhibits FGF19-induced repression of CYP7α1 gene in a cell. 12.The method of inhibiting relapse tumor growth of claim 1, wherein theantibody inhibits FGF19-induced phosphorylation of one or more of FGFR4,MAPK, FRS and ERK2.
 13. The method of inhibiting relapse tumor growth ofclaim 1, wherein the antibody inhibits Wnt pathway activation in a cell.14. The method of inhibiting relapse tumor growth of claim 13, whereinWnt pathway activation is characterized by one or more of tyrosinephosphorylation of β-catenin, expression of Wnt target genes, andE-cadherin binding to β-catenin.
 15. The method of inhibiting relapsetumor growth of claim 1, wherein the antibody is a monoclonal antibody.16. The method of inhibiting relapse tumor growth of claim 1, whereinthe antibody is selected from the group consisting of a chimericantibody, a humanized antibody, an affinity matured antibody, a humanantibody, and a bispecific antibody.
 17. The method of inhibitingrelapse tumor growth of claim 1, wherein the antibody is an antibodyfragment.
 18. The method of inhibiting relapse tumor growth claim 1,wherein the antibody is an immunoconjugate.